Methods For The Diagnosis Of Fetal Abnormalities

ABSTRACT

The present invention relates to methods for detecting, enriching, and analyzing rare cells that are present in the blood, e.g. fetal cells. The invention further features methods of analyzing rare cell(s) to determine the presence of an abnormality, disease or condition in a subject, e.g. a fetus by analyzing a cellular sample from the subject.

CROSS-REFERENCE

This application claims the benefit of U.S. Provisional Application No.60/804,817, filed Jun. 14, 2006, which application is incorporatedherein by reference.

BACKGROUND OF THE INVENTION

Analysis of specific cells can give insight into a variety of diseases.These analyses can provide non-invasive tests for detection, diagnosisand prognosis of diseases, thereby eliminating the risk of invasivediagnosis. For instance, social developments have resulted in anincreased number of prenatal tests. However, the available methodstoday, amniocentesis and chorionic villas sampling (CVS) are potentiallyharmful to the mother and to the fetus. The rate of miscarriage forpregnant women undergoing amniocentesis is increased by 0.5-1%, and thatfigure is slightly higher for CVS. Because of the inherent risks posedby amniocentesis and CVS, these procedures are offered primarily toolder women, i.e., those over 35 years of age, who have a statisticallygreater probability of bearing children with congenital defects. As aresult, a pregnant woman at the age of 35 has to balance an average riskof 0.5-1% to induce an abortion by amniocentesis against an age relatedprobability for trisomy 21 of less than 0.3%. To eliminate the risksassociated with invasive prenatal screening procedures, non-invasivetests for detection, diagnosis and prognosis of diseases, have beenutilized. For example, maternal serum alpha-fetoprotein, and levels ofunconjugated estriol and human chorionic gonadotropin are used toidentify a proportion of fetuses with Down's syndrome, however, thesetests are not one hundred percent accurate. Similarly, ultrasonographyis used to determine congenital defects involving neural tube defectsand limb abnormalities, but is useful only after fifteen weeks'gestation

Moreover, despite decades of advances in cancer diagnosis and therapy,many cancers continue to go undetected until late in their development.As one example, most early-stage lung cancers are asymptomatic and arenot detected in time for curative treatment, resulting in an overallfive-year survival rate for patients with lung cancer of less than 15%.However, in those instances in which lung cancer is detected and treatedat an early stage, the prognosis is much more favorable.

The presence of fetal cells in the maternal circulation and cancer cellsin patients' circulation offers an opportunity to develop prenataldiagnostics that obviates the risks associated with invasive diagnosticprocedure, and cancer diagnostics that allow for detecting cancer atearlier stages in the development of the disease. However, fetal cellsand cancer cells are rare as compared to the presence of other cells inthe blood. Therefore, any proposed analysis of fetal cells or cancercells to diagnose fetal abnormalities or cancers, respectively, requiresenrichment of fetal cells and cancer cells. Enriching fetal cells frommaternal peripheral blood and cancer cells from patient's blood ischallenging, time intensive and any analysis derived there from is proneto error. The present invention addresses these challenges.

SUMMARY OF THE INVENTION

The methods of the present invention allow for enrichment of rare cellpopulations, particularly fetal cells or cancer cells, from peripheralblood samples which enrichment yields cell populations sufficient forreliable and accurate clinical diagnosis. The methods of the presentinvention also provide analysis of said enriched rare cell populationswhereby said methods allow for detection, diagnosis and prognosis ofconditions or diseases, in particular fetal abnormalities or cancer.

The present invention relates to methods for determining a condition ina patient or a fetus by analyzing nucleic acids from cells of samplesobtained from patient or maternal samples, respectively. The methodsinclude enriching the sample for cells that are normally present in vivoat a concentration of less than 1 in 100,000, obtaining the nuclei fromthe enriched sample cells and detecting substantially in real time oneor more nucleic acids molecules. The sample can be enriched for avariety of cells including fetal cells, epithelial cells, endothelialcells or progenitor cells, and the sample can be obtained from a varietyof sources including whole blood, sweat, tears, ear flow, sputum, lymph,bone marrow suspension, lymph, urine, saliva, semen, vaginal flow,cerebrospinal fluid, brain fluid, ascites, milk, secretions of therespiratory, intestinal or genitourinary tracts fluid. Preferably, thesample is a blood sample.

In some embodiments, samples are enriched in fetal cells, and thecondition that can be determined by the methods of the invention can bea genetic or pathologic condition. In some embodiments, geneticconditions that can be determined in one or more fetal cells includetrisomy 13, trisomy 18, trisomy 21, Klinefelter Syndrome,dup(17)(p11.2p11.2) syndrome, Down syndrome, Pre-eclampsia, Pre-termlabor, Edometriosis, Pelizaeus-Merzbacher disease, dup(22)(q11.2q11.2)syndrome, Cat eye syndrome, Cri-du-chat syndrome, Wolf-Hirschhornsyndrome, Williams-Beuren syndrome, Charcot-Marie-Tooth disease,neuropathy with liability to pressure palsies, Smith-Magenis syndrome,neurofibromatosis, Alagille syndrome, Velocardiofacial syndrome,DiGeorge syndrome, steroid sulfatase deficiency, Kallmann syndrome,microphthalmia with linear skin defects, Adrenal hypoplasia, Glycerolkinase deficiency, Pelizaeus-Merzbacher disease, testis-determiningfactor on Y, Azospermia (factor a), Azospermia (factor b), Azospermia(factor c), or 1p36 deletion. In other embodiments, the P conditionsthat can be determined in one or more fetal cells include acutelymphoblastic leukemia, acute or chronic lymphocyctic or granulocytictumor, acute myeloid leukemia, acute promyelocytic leukemia,adenocarcinoma, adenoma, adrenal cancer, basal cell carcinoma, bonecancer, brain cancer, breast cancer, bronchi cancer, cervical dysplasia,chronic myelogenous leukemia, colon cancer, epidermoid carcinoma,Ewing's sarcoma, gallbladder cancer, gallstone tumor, giant cell tumor,glioblastoma multiforma, hairy-cell tumor, head cancer, hyperplasia,hyperplastic conical nerve tumor, in situ carcinoma, intestinalganglioneuroma, islet cell tumor, Kaposi's sarcoma, kidney cancer,larynx cancer, leiomyomater tumor, liver cancer, lung cancer, lymphomas,malignant carcinoid, malignant hypercalcemia, malignant melanomas,marfanoid habitus tumor, medullary carcinoma, metastatic skin carcinoma,mucosal neuromas, mycosis fungoide, myelodysplastic syndrome, myeloma,neck cancer, neural tissue cancer, neuroblastoma, osteogenic sarcoma,osteosarcoma, ovarian tumor, pancreas cancer, parathyroid cancer,pheochromocytoma, polycythemia vera, primary brain tumor, prostatecancer, rectum cancer, renal cell tumor, retinoblastoma,rhabdomyosarcoma, seminoma, skin cancer, small-cell lung tumor, softtissue sarcoma, squamous cell carcinoma, stomach cancer, thyroid cancer,topical skin lesion, veticulum cell sarcoma, or Wilm's tumor.

In some embodiments, the step of enriching a sample for a cell typeincludes flowing a sample or a fraction of a sample through an array ofobstacles that separate the cells according to size by selectivelydirecting cells of a predetermined size into a first outlet anddirecting cells of another predetermined size to a second outlet, andflowing the sample or sample fraction through one or more magneticfields that retain paramagnetic components. The method further comprisesejecting the nuclei from the cells in the sample by applying hyperbaricor hypobaric pressure to the sample, and flowing the sample or a samplefraction through an array of obstacles that are coated with bindingmoieties that bind one or more cell populations in the sample. Thebinding moieties of this and other disclosed methods can be any known inthe art and may be selected from the group consisting of antibodies,receptors, ligands, proteins, nucleic acids, sugars, carbohydrates andcombinations thereof.

In some embodiments, the methods of the invention can be used todetermine a fetal abnormality from amniotic fluid obtained from apregnant female. In these embodiments, an amniotic fluid sample isobtained from the pregnant female and is enriched for fetal cells.Subsequently, one or more nucleic acid molecules are obtained from theenriched cells, and are amplified on a bead. Up to 100 bases of thenucleic acid are obtained, and in some embodiments up to one millioncopies of the nucleic acid are amplified. The amplified nucleic acidscan also be sequenced. Preferably, the nucleic acid is genomic DNA.

In some embodiments, the fetal abnormality can be determined from asample that is obtained from a pregnant female and enriched for fetalcells by subjecting the sample to the enrichment procedure that includesseparating cells according size, and flowing it through a magneticfield. The size-based separation involves flowing the sample through anarray of obstacles that directs cells of a size smaller than apredetermined size to a first outlet, and cells that are larger than apredetermined size to a second outlet. The enriched sample is alsosubjected to one or more magnetic fields and hyperbaric or hypobaricpressure, and in some embodiments it is used for genetic analysesincluding SNP detection, RNA expression detection and sequencedetection. In some embodiments, one or more nucleic acid fragments canbe obtained from the sample that has been subjected to the hyperbaric orhypobaric pressure, and the nucleic acid fragments can be amplified bymethods including multiple displacement amplification (MDA), degenerateoligonucleotide primed PCR (DOP), primer extension pre-amplification(PEP) or improved-PEP (1-PEP).

In some embodiments, the method for determining a fetal abnormality canbe performed using a blood sample obtained form a pregnant female. Thesample can be enriched for fetal cells by flowing the sample through anarray of obstacles that directs cells of a size smaller than apredetermined size to a first outlet, and cells that are larger than apredetermined size to a second outlet, and performing a genetic analysise.g. SNP detection, RNA expression detection and sequence detection, onthe enriched sample. The enriched sample can comprise one or more fetalcells and one or more nonfetal cells.

In some embodiments the invention includes kits providing the devicesand reagents for performing one or all of the steps for determining thefetal abnormalities. These kits may include any of the devices orreagents disclosed singly or in combination.

In some embodiments, the genetic analysis of SNP detection or RNAexpression can be performed using microarrays. SNP detection can also beaccomplished using molecular inverted probes(s), and in someembodiments, SNP detection involves highly parallel detection of atleast 100,000 SNPs. RNA expression detection can also involve highlyparallel analysis of at least 10,000 transcripts. In some embodiments,sequence detection can involve determining the sequence of at least50,000 bases per hour, and sequencing can be done in substantially realtime or real time and can comprise adding a plurality of labelednucleotides or nucleotide analogs to a sequence that is complementary tothat of the enriched nucleic acid molecules, and detecting theincorporation. A variety of labels can be used in the sequence detectionstep and include chromophores, fluorescent moieties, enzymes, antigens,heavy metal, magnetic probes, dyes, phosphorescent groups, radioactivematerials, chemiluminescent moieties, scattering or fluorescentnanoparticles, Raman signal generating moieties, and electrochemicaldetection moieties. Methods that include sequence detection can beaccomplished using sequence by synthesis and they may include amplifyingthe nucleic acid on a bead. In some embodiments, the methods can includeamplifying target nucleic acids from the enriched sample(s) by anymethod known in the art but preferably by multiple displacementamplification (MDA), degenerate oligonucleotide primed PCR (DOP), primerextension pre-amplification (PEP) or improved-PEP (1-PEP).

The genetic analyses can be performed on DNA of chromosomes X, Y, 13, 18or 21 or on the RNA transcribed therefrom. In some embodiments, thegenetic analyses can also be performed on a control sample or referencesample, and in some instances, the control sample can be a maternalsample.

SUMMARY OF THE DRAWINGS

FIGS. 1A-1D illustrate embodiments of a size-based separation module.

FIGS. 2A-2C illustrate one embodiment of an affinity separation module.

FIG. 3 illustrate one embodiment of a magnetic separation module.

FIG. 4 illustrates one example of a multiplex enrichment module of thepresent invention.

FIG. 5 illustrates exemplary genes that can be analyzed from enrichedcells, such as epithelial cells, endothelial cells, circulating tumorcells, progenitor cells, etc.

FIG. 6 illustrates one embodiment for genotyping rare cell(s) or rareDNA using, e.g., Affymetrix DNA microarrays.

FIG. 7 illustrates one embodiment for genotyping rare cell(s) or rareDNA using, e.g., Illumina bead arrays.

FIG. 8 illustrates one embodiment for determining gene expression ofrare cell(s) or rare DNA using, e.g., Affymetrix expression chips.

FIG. 9 illustrates one embodiment for determining gene expression ofrare cell(s) or rare DNA using, e.g., Illumina bead arrays.

FIG. 10 illustrates one embodiment for high-throughput sequencing ofrare cell(s) or rare DNA using, e.g., single molecule sequence bysynthesis methods (e.g., Helicos BioSciences Corporation).

FIG. 11 illustrates one embodiment for high-throughput sequencing ofrare cell(s) or rare DNA using, e.g., amplification of nucleic acidmolecules on a bead (e.g., 454 Lifesciences).

FIG. 12 illustrates one embodiment for high-throughput sequencing ofrare cell(s) or rare DNA using, e.g., clonal single molecule arraystechnology (e.g., Solexa, Inc.).

FIG. 13 illustrates one embodiment for high-throughput sequencing ofrare cell(s) or rare DNA using, e.g., single base polymerization usingenhanced nucleotide fluorescence (e.g., Genovoxx GmbH).

FIGS. 14A-14D illustrate one embodiment of a device used to separatecells according to their size.

FIGS. 15A-15B illustrate cell smears of first and second outlet (e.g.,product and waste) fractions.

FIGS. 16A-16F illustrate isolation of CD-71 positive population from anucleated cell fraction.

FIG. 17 illustrates trisomy 21 pathology.

FIG. 18 illustrates performance of cell separation module.

FIG. 19 illustrates histograms representative of cell fractionsresulting from cell separation module described herein.

FIG. 20 illustrates cytology of products from cell separation module.

FIG. 21 illustrates epithelial cells bound to obstacles and floor in aseparation/enrichment module.

FIG. 22 illustrates a process for analyzing enriched epithelial cellsfor EGFR mutations.

FIG. 23 illustrates a method for generating sequences templates forregions of interest.

FIG. 24 illustrates exemplary allele specific reactions showingmutations.

FIG. 25 illustrates exemplary signals from an ABT.

FIG. 26A illustrates BCKDK expressed in leukocytes and H1650 cells.

FIG. 26B illustrates EGFR expression profile.

FIG. 27 illustrates the detection of single copies of a fetal cellgenome by qPCR.

FIG. 28 illustrates detection of single fetal cells in binned samples bySNP analysis.

FIG. 29 illustrates a method of trisomy testing. The trisomy 21 screenis based on scoring of target cells obtained from maternal blood. Bloodis processed using a cell separation module for hemoglobin enrichment(CSM-HE). Isolated cells are transferred to slides that are firststained and subsequently probed by FISH. Images are acquired, such asfrom bright field or fluorescent microscopy, and scored. The proportionof trisomic cells of certain classes serves as a classifier for risk offetal trisomy 21. Fetal genome identification can performed using assayssuch as: (1) STR markers; (2) qPCR using primers and probes directed toloci, such as the multi-repeat DYZ locus on the Y-chromosome; (3) SNPdetection; and (4) CGH (comparative genome hybridization) arraydetection.

FIG. 30 illustrates assays that can produce information on the presenceof aneuploidy and other genetic disorders in target cells. Informationon aneuploidy and other genetic disorders in target cells may beacquired using technologies such as: (1) a CGH array established forchromosome counting, which can be used for aneuploidy determinationand/or detection of intra-chromosomal deletions; (2) SNP/taqman assays,which can be used for detection of single nucleotide polymorphisms; and(3) ultra-deep sequencing, which can be used to produce partial orcomplete genome sequences for analysis.

FIG. 31 illustrates methods of fetal diagnostic assays. Fetal cells areisolated by CSM-HE enrichment of target cells from blood. Thedesignation of the fetal cells may be confirmed using techniquescomprising FISH staining (using slides or membranes and optionally anautomated detector), FACS, and/or binning. Binning may comprisedistribution of enriched cells across wells in a plate (such as a 96 or384 well plate), microencapsulation of cells in droplets that areseparated in an emulsion, or by introduction of cells into microarraysof nanofluidic bins. Fetal cells are then identified using methods thatmay comprise the use of biomarkers (such as fetal (gamma) hemoglobin),allele-specific SNP panels that could detect fetal genome DNA, detectionof differentially expressed maternal and fetal transcripts (such asAffymetrix chips), or primers and probes directed to fetal specific loci(such as the multi-repeat DYZ locus on the Y-chromosome). Binning sitesthat contain fetal cells are then be analyzed for aneuploidy and/orother genetic defects using a technique such as CGH array detection,ultra deep sequencing (such as Solexa, 454, or mass spectrometry), STRanalysis, or SNP detection.

FIG. 32 illustrates methods of fetal diagnostic assays, furthercomprising the step of whole genome amplification prior to analysis ofaneuploidy and/or other genetic defects.

INCORPORATION BY REFERENCE

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides systems, apparatus, and methods to detectthe presence of or abnormalities of rare analytes or cells, such ashematapoeitic bone marrow pregenetor cells, endothelial cells, fetalcells circulating in maternal peripheral blood, epithelial cells, orcirculating tumor cells in a sample of a mixed analyte or cellpopulation (e.g. maternal peripheral blood samples).

1. Sample Collection/Preparation

Samples containing rare cells can be obtained from any animal in need ofa diagnosis or prognosis or from an animal pregnant with a fetus in needof a diagnosis or prognosis. In one example, a sample can be obtainedfrom animal suspected of being pregnant, pregnant, or that has beenpregnant to detect the presence of a fetus or fetal abnormality. Inanother example, a sample is obtained from an animal suspected ofhaving, having, or an animal that had a disease or condition (e.g.cancer). Such condition can be diagnosed, prognosed, monitored andtherapy can be determined based on the methods and systems herein.Animal of the present invention can be a human or a domesticated animalsuch as a cow, chicken, pig, horse, rabbit, dogs, cat, or goat. Samplesderived from an animal or human can include, e.g., whole blood, sweat,tears, ear flow, sputum, lymph, bone marrow suspension, lymph, urine,saliva, semen, vaginal flow, cerebrospinal fluid, brain fluid, ascites,milk, secretions of the respiratory, intestinal or genitourinary tractsfluid.

To obtain a blood sample, any technique known in the art may be used,e.g. a syringe or other vacuum suction device. A blood sample can beoptionally pre-treated or processed prior to enrichment. Examples ofpre-treatment steps include the addition of a reagent such as astabilizer, a preservative, a fixant, a lysing reagent, a diluent, ananti-apoptotic reagent, an anti-coagulation reagent, an anti-thromboticreagent, magnetic property regulating reagent, a buffering reagent, anosmolality regulating reagent, a pH regulating reagent, and/or across-linking reagent.

When a blood sample is obtained, a preservative such an anti-coagulationagent and/or a stabilizer is often added to the sample prior toenrichment. This allows for extended time for analysis/detection. Thus,a sample, such as a blood sample, can be enriched and/or analyzed underany of the methods and systems herein within 1 week, 6 days, 5 days, 4days, 3 days, 2 days, 1 day, 12 hrs, 6 hrs, 3 hrs, 2 hrs, or 1 hr fromthe time the sample is obtained.

In some embodiments, a blood sample can be combined with an agent thatselectively lyses one or more cells or components in a blood sample. Forexample, fetal cells can be selectively lysed releasing their nucleiwhen a blood sample including fetal cells is combined with deionizedwater. Such selective lysis allows for the subsequent enrichment offetal nuclei using, e.g., size or affinity based separation. In anotherexample platelets and/or enucleated red blood cells are selectivelylysed to generate a sample enriched in nucleated cells, such as fetalnucleated red blood cells (fnRBC's), maternal nucleated blood cells(mnBC), epithelial cells and circulating tumor cells. fnRBC's can besubsequently separated from mnBC's using, e.g., antigen-i affinity ordifferences in hemoglobin

When obtaining a sample from an animal (e.g., blood sample), the amountcan vary depending upon animal size, its gestation period, and thecondition being screened. In some embodiments, up to 50, 40, 30, 20, 10,9, 8, 7, 6, 5, 4, 3, 2, or 1 mL of a sample is obtained. In someembodiments, 1-50, 2-40, 3-30, or 4-20 mL of sample is obtained. In someembodiments, more than 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60,65, 70, 75, 80, 85, 90, 95 or 100 mL of a sample is obtained.

To detect fetal abnormality, a blood sample can be obtained from apregnant animal or human within 36, 24, 22, 20, 18, 16, 14, 12, 10, 8, 6or 4 weeks of gestation or even after a pregnancy has terminated.

II. Enrichment

A sample (e.g. blood sample) can be enriched for rare analytes or rarecells (e.g. fetal cells, epithelial cells or circulating tumor cells)using one or more any methods known in the art (e.g. Guetta, E M et al.Stem Cells Dev, 13(1):93-9 (2004)) or described herein. The enrichmentincreases the concentration of rare cells or ratio of rare cells tonon-rare cells in the sample. For example, enrichment'can increaseconcentration of an analyte of interest such as a fetal cell orepithelial cell or circulating tumor cell (CTC) by a factor of at least2, 4, 6, 8, 10, 20, 50, 100, 200, 500, 1,000, 2,000, 5,000, 10,000,20,000, 50,000, 100,000, 200,000, 500,000, 1,000,000, 2,000,000,5,000,000, 10,000,000, 20,000,000, 50,000,000, 100,000,000, 200,000,000,500,000,000, 1,000,000,000, 2,000,000,000, or 5,000,000,000 fold overits concentration in the original sample. In particular, when enrichingfetal cells from a maternal peripheral venous blood sample, the initialconcentration of the fetal cells may be about 1:50,000,000 and it may beincreased to at least 1:5,000 or 1:500. Enrichment can also increaseconcentration of rare cells in volume of rare cells/total volume ofsample (removal of fluid). A fluid sample (e.g., a blood sample) ofgreater than 10, 15, 20, 50, or 100 mL total volume comprising rarecomponents of interest, and it can be concentrated such that the rarecomponent of interest into a concentrated solution of less than 0.5, 1,2, 3, 5, or 10 mL total volume.

Enrichment can occur using one or more types of separation modules.Several different modules are described herein, all of which can befluidly coupled with one another in the series for enhanced performance.

In some embodiments, enrichment occurs by selective lysis as describedabove.

In one embodiment, enrichment of rare cells occurs using one or moresize-based separation modules. Examples of size-based separation modulesinclude filtration modules, sieves, matrixes, etc. Examples ofsize-based separation modules contemplated by the present inventioninclude those disclosed in International Publication. No. WO2004/113877. Other size based separation modules are disclosed inInternational Publication No. WO 2004/0144651.

In some embodiments, a size-based separation module comprises one ormore arrays of obstacles forming a network of gaps. The obstacles areconfigured to direct particles as they flow through the array/network ofgaps into different directions or outlets based on the particle'shydrodynamic size. For example, as a blood sample flows through an arrayof obstacles, nucleated cells or cells having a hydrodynamic size largerthan a predetermined size, e.g., 8 microns, are directed to a firstoutlet located on the opposite side of the array of obstacles from thefluid flow inlet, while the enucleated cells or cells having ahydrodynamic size smaller than a predetermined size, e.g., 8 microns,are directed to a second outlet also located on the opposite side of thearray of obstacles from the fluid flow inlet.

An array can be configured to separate cells smaller or larger than apredetermined size by adjusting the size of the gaps, obstacles, andoffset in the period between each successive row of obstacles. Forexample, in some embodiments, obstacles or gaps between obstacles can beup to 10, 20, 50, 70, 100, 120, 150, 170, or 200 microns in length orabout 2, 4, 6, 8 or 10 microns in length. In some embodiments, an arrayfor size-based separation includes more than 100, 500, 1,000, 5,000,10,000, 50,000 or 100,000 obstacles that are arranged into more than 10,20, 50, 100, 200, 500, or 1000 rows. Preferably, obstacles in a firstrow of obstacles are offset from a previous (upstream) row of obstaclesby up to 50% the period of the previous row of obstacles. In someembodiments, obstacles in a first row of obstacles are offset from aprevious row of obstacles by up to 45, 40, 35, 30, 25, 20, 15 or 10% theperiod of the previous row of obstacles. Furthermore, the distancebetween a first row of obstacles and a second row of obstacles can be upto 10, 20, 50, 70, 100, 120, 150, 170 or 200 microns. A particularoffset can be continuous (repeating for multiple rows) ornon-continuous. In some embodiments, a separation module includesmultiple discrete arrays of obstacles fluidly coupled such that they arein series with one another. Each array of obstacles has a continuousoffset. But each subsequent (downstream) array of obstacles has anoffset that is different from the previous (upstream) offset.Preferably, each subsequent array of obstacles has a smaller offset thatthe previous array of obstacles. This allows for a refinement in theseparation process as cells migrate through the array of obstacles.Thus, a plurality of arrays can be fluidly coupled in series or inparallel, (e.g., more than 2, 4, 6, 8, 10, 20, 30, 40, 50). Fluidlycoupling separation modules (e.g., arrays) in parallel allows forhigh-throughput analysis of the sample, such that at least 1, 2, 5, 10,20, 50, 100, 200, or 500 mL per hour flows through the enrichmentmodules or at least 1, 5, 10, or 50 million cells per hour are sorted orflow through the device.

FIG. 1A illustrates an example of a size-based separation module.Obstacles (which may be of any shape) are coupled to a flat substrate toform an array of gaps. A transparent cover or lid may be used to coverthe array. The obstacles form a two-dimensional array with eachsuccessive row shifted horizontally with respect to the previous row ofobstacles, where the array of obstacles directs component having ahydrodynamic size smaller than a predetermined size in a first directionand component having a hydrodynamic size larger that a predeterminedsize in a second direction. For enriching epithelial or circulatingtumor cells from enucleated, the predetermined size of an array ofobstacles can be get at 6-12 μm or 6-8 μm. For enriching fetal cellsfrom a mixed sample (e.g. maternal blood sample) the predetermined sizeof an array of obstacles can be get at between 4-10 μm or 6-8 μm. Theflow of sample into the array of obstacles can be aligned at a smallangle (flow angle) with respect to a line-of-sight of the array.Optionally, the array is coupled to an infusion pump to perfuse thesample through the obstacles. The flow conditions of the size-basedseparation module described herein are such that cells are sorted by thearray with minimal damage. This allows for downstream analysis of intactcells and intact nuclei to be more efficient and reliable.

In some embodiments, a size-based separation module comprises an arrayof obstacles configured to direct cells larger than a predetermined sizeto migrate along a line-of-sight within the array (e.g. towards a firstoutlet or bypass channel leading to a first outlet), while directingcells and analytes smaller than a predetermined size to migrate throughthe array of obstacles in a different direction than the larger cells(e.g. towards a second outlet). Such embodiments are illustrated in partin FIGS. 1B-1D.

A variety of enrichment protocols may be utilized although gentlehandling of the cells is preferred to reduce any mechanical damage tothe cells or their DNA. This gentle handling may serve to preserve thesmall number of fetal cells in the sample. Integrity of the nucleic acidbeing evaluated is an important feature to permit the distinctionbetween the genomic material from the fetal cells and other cells in thesample. In particular, the enrichment and separation of the fetal cellsusing the arrays of obstacles produces gentle treatment which minimizescellular damage and maximizes nucleic acid integrity permittingexceptional levels of separation and the ability to subsequently utilizevarious formats to very accurately analyze the genome of the cells whichare present in the sample in extremely low numbers.

In some embodiments, enrichment of rare cells (e.g. fetal cells,epithelial cells or circulating tumor cells) occurs using one or morecapture modules that selectively inhibit the mobility of one or morecells of interest. Preferably, a capture module is fluidly coupleddownstream to a size-based separation module. Capture modules caninclude a substrate having multiple obstacles that restrict the movementof cells or analytes greater than a predetermined size. Examples ofcapture modules that inhibit the migration of cells based on size aredisclosed in U.S. Pat. Nos. 5,837,115 and 6,692,952.

In some embodiments, a capture module includes a two dimensional arrayof obstacles that selectively filters or captures cells or analyteshaving a hydrodynamic size greater than a particular gap size(predetermined size), International Publication No. WO 2004/113877.

In some cases a capture module captures analytes (e.g., cells ofinterest or not of interest) based on their affinity. For example, anaffinity-based separation module that can capture cells or analytes caninclude an array of obstacles adapted for permitting sample flowthrough, but for the fact that the obstacles are covered with bindingmoieties that selectively bind one or more analytes (e.g., cellpopulations) of interest (e.g., red blood cells, fetal cells, epithelialcells or nucleated cells) or analytes not-of-interest (e.g., white bloodcells). Arrays of obstacles adapted for separation by capture caninclude obstacles having one or more shapes and can be arranged in auniform or non-uniform order. In some embodiments, a two-dimensionalarray of obstacles is staggered such that each subsequent row ofobstacles is offset from the previous row of obstacles to increase thenumber of interactions between the analytes being sorted (separated) andthe obstacles.

Binding moieties coupled to the obstacles can include e.g., proteins(e.g., ligands/receptors), nucleic acids having complementarycounterparts in retained analytes, antibodies, etc. In some embodiments,an affinity-based separation module comprises a two-dimensional array ofobstacles covered with one or more antibodies selected from the groupconsisting of anti-CD71, anti-CD235a, anti-CD36, anti-carbohydrates,anti-selectin, anti-CD45, anti-GPA, anti-antigen-i, anti-EpCAM,anti-E-cadherin, and anti-Muc-1.

FIG. 2A illustrates a path of a first analyte through an array of postswherein an analyte that does not specifically bind to a post continuesto migrate through the array, while an analyte that does bind a post iscaptured by the array. FIG. 2B is a picture of antibody coated posts.FIG. 2C illustrates coupling of antibodies to a substrate (e.g.,obstacles, side walls, etc.) as contemplated by the present invention.Examples of such affinity-based separation modules are described inInternational Publication No. WO 2004/029221.

In some embodiments, a capture module utilizes a magnetic field toseparate and/or enrich one or more analytes (cells) based on a magneticproperty or magnetic potential in such analyte of interest or an analytenot of interest. For example, red blood cells which are slightlydiamagnetic (repelled by magnetic field) in physiological conditions canbe made paramagnetic (attributed by magnetic field) by deoxygenation ofthe hemoglobin into methemoglobin. This magnetic property can beachieved through physical or chemical treatment of the red blood cells.Thus, a sample containing one or more red blood cells and one or morewhite blood cells can be enriched for the red blood cells by firstinducing a magnetic property in the red blood cells and then separatingthe red blood cells from the white blood cells by flowing the samplethrough a magnetic field (uniform or non-uniform).

For example, a maternal blood sample can flow first through a size-basedseparation module to remove enucleated cells and cellular components(e.g., analytes having a hydrodynamic size less than 6 μms) based onsize. Subsequently, the enriched nucleated cells (e.g., analytes havinga hydrodynamic size greater than 6 μms) white blood cells and nucleatedred blood cells are treated with a reagent, such as CO₂, N₂, or NaNO₂,that changes the magnetic property of the red blood cells' hemoglobin.The treated sample then flows through a magnetic field (e.g., a columncoupled to an external magnet), such that the paramagnetic analytes(e.g., red blood cells) will be captured by the magnetic field while thewhite blood cells and any other non-red blood cells will flow throughthe device to result in a sample enriched in nucleated red blood cells(including fetal nucleated red blood cells or fnRBC's). Additionalexamples of magnetic separation modules are described in U.S.application Ser. No. 11/323,971, filed Dec. 29, 2005 entitled “Devicesand Methods for Magnetic Enrichment of Cells and Other Particles” andU.S. application Ser. No. 11/227,904, filed Sep. 15, 2005, entitled“Devices and Methods for Enrichment and Alteration of Cells and OtherParticles”.

Subsequent enrichment steps can be used to separate the rare cells (e.g.fnRBC's) from the non-rare cells maternal nucleated red blood cells. Insome embodiments, a sample enriched by size-based separation followed byaffinity/magnetic separation is further enriched for rare cells usingfluorescence activated cell sorting (FACS) or selective lysis of asubset of the cells.

In some embodiments, enrichment involves detection and/or isolation ofrare cells or rare DNA (e.g. fetal cells or fetal DNA) by selectivelyinitiating apoptosis in the rare cells. This can be accomplished, forexample, by subjecting a sample that includes rare cells (e.g. a mixedsample) to hyperbaric pressure (increased levels of CO₂; e.g. 4% CO₂).This will selectively initiate condensation and/or apoptosis in the rareor fragile cells in the sample (e.g. fetal cells). Once the rare cells(e.g. fetal cells) begin apoptosis, their nuclei will condense andoptionally be ejected from the rare cells. At that point, the rare cellsor nuclei can be detected using any technique known in the art to detectcondensed nuclei, including DNA gel electropheresis, in situ labelingfluorescence labeling, and in situ labeling of DNA nicks using terminaldeoxynucleotidyl transferase (TdT)-mediated dUTP in situ nick labeling(TUNEL) (Gavrieli, Y., et al. J. Cell. Biol. 119:493-501 (1992)), andligation of DNA strand breaks having one or two-base 3′ overhangs (Taqpolymerase-based in situ ligation). (Didenko V., et al. J. Cell Biol.135:1369-76 (1996)).

In some embodiments ejected nuclei can further be detected using a sizebased separation module adapted to selectively enrich nuclei and otheranalytes smaller than a predetermined size (e.g. 6 microns) and isolatethem from cells and analytes having a hydrodynamic diameter larger than6 microns. Thus, in one embodiment, the present invention contemplateddetecting fetal cells/fetal. DNA and optionally using such fetal DNA todiagnose or prognose a condition in a fetus. Such detection anddiagnosis can occur by obtaining a blood sample from the female pregnantwith the fetus, enriching the sample for cells and analytes larger than8 microns using, for example, an array of obstacles adapted forsize-base separation where the predetermined size of the separation is 8microns (e.g. the gap between obstacles is up to 8 microns). Then, theenriched product is further enriched for red blood cells (RBC's) byoxidizing the sample to make the hemaglobin paramagnetic and flowing thesample through one or more magnetic regions. This selectively capturesthe RBC's and removes other cells (e.g. white blood cells) from thesample. Subsequently, the fnRBC's can be enriched from mnRBC's in thesecond enriched product by subjecting the second enriched product tohyperbaric or hypobaric pressure or other stimulus that selectivelycauses the fetal cells to begin apoptosis and condense/eject theirnuclei. Such condensed nuclei are then identified/isolated using e.g.laser capture microdissection or a size based separation module thatseparates components smaller than 3, 4, 5 or 6 microns from a sample.Such fetal nuclei can then by analyzed using any method known in the artor described herein.

In some embodiments, when the analyte desired to be separated (e.g., redblood cells or white blood cells) is not ferromagnetic or does not havea potential magnetic property, a magnetic particle (e.g., a bead) orcompound (e.g., Fe³⁺) can be coupled to the analyte to give it amagnetic property. In some embodiments, a bead coupled to an antibodythat selectively binds to an analyte of interest can be decorated withan antibody elected from the group of anti CD71 or CD75. In someembodiments a magnetic compound, such as Fe³⁺, can be couple to anantibody such as those described above. The magnetic particles ormagnetic antibodies herein may be coupled to any one or more of thedevices herein prior to contact with a sample or may be mixed with thesample prior to delivery of the sample to the device(s). Magneticparticles can also be used to decorate one or more analytes (cells ofinterest or not of interest) to increase the size prior to performingsize-based separation.

Magnetic field used to separate analytes/cells in any of the embodimentsherein can uniform or non-uniform as well as external or internal to thedevice(s) herein. An external magnetic field is one whose source isoutside a device herein (e.g., container, channel, obstacles). Aninternal magnetic field is one whose source is within a devicecontemplated herein. An example of an internal magnetic field is onewhere magnetic particles may be attached to obstacles present in thedevice (or manipulated to create obstacles) to increase surface area foranalytes to interact with to increase the likelihood of binding.Analytes captured by a magnetic field can be released by demagnetizingthe magnetic regions retaining the magnetic particles. For selectiverelease of analytes from regions, the demagnetization can be limited toselected obstacles or regions. For example, the magnetic field can bedesigned to be electromagnetic, enabling turn-on and turn-off off themagnetic fields for each individual region or obstacle at will.

FIG. 3 illustrates an embodiment of a device configured for capture andisolation of cells expressing the transferrin receptor from a complexmixture. Monoclonal antibodies to CD71 receptor are readily availableoff-the-shelf and can be covalently coupled to magnetic materials, suchas, but not limited to any ferroparticles including but not limited toferrous doped polystyrene and ferroparticles or Ferro-colloids (e.g.,from Miltenyi and Dynal). The anti CD71 bound to magnetic particles isflowed into the device. The antibody coated particles are drawn to theobstacles (e.g., posts), floor, and walls and are retained by thestrength of the magnetic field interaction between the particles and themagnetic field. The particles between the obstacles and those looselyretained with the sphere of influence of the local magnetic fields awayfrom the obstacles are removed by a rinse.

In some cases, a fluid sample such as a blood sample is first flowedthrough one or more size-base separation module. Such modules may befluidly connected in series and/or in parallel. FIG. 4 illustrates oneembodiment of three size-based enrichment modules that are fluidlycoupled in parallel. The waste (e.g., cells having hydrodynamic sizeless than 4 microns) are directed into a first outlet and the product(e.g., cells having hydrodynamic size greater than 4 microns) aredirected to a second outlet. The product is subsequently enriched usingthe inherent magentic property of hemoglobin. The product is modified(e.g., by addition of one or more reagents) such that the hemoglobin inthe red blood cells becomes paramagentic. Subsequently, the product isflowed through one or more magentic fields. The cells that are trappedby the magentic field are subsequently analyzed using the one or moremethods herein.

One or more of the enrichment modules herein (e.g., size-basedseparation module(s) and capture module(s)) may be fluidly coupled inseries or in parallel with one another. For example a first outlet froma separation module can be fluidly coupled to a capture module. In someembodiments, the separation module and capture module are integratedsuch that a plurality of obstacles acts both to deflect certain analytesaccording to size and direct them in a path different than the directionof analyte(s) of interest, and also as a capture module to capture,retain, or bind certain analytes based on size, affinity, magnetism orother physical property.

In any of the embodiments herein, the enrichment steps performed have aspecificity and/or sensitivity greater than 50, 60, 70, 80, 90, 95, 96,97, 98, 99, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, 99.9 or99.95% The retention rate of the enrichment module(s) herein is suchthat ≧50, 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 99.9%of the analytes or cells of interest (e.g., nucleated cells or nucleatedred blood cells or nucleated from red blood cells) are retained.Simultaneously, the enrichment modules are configured to remove ≧50, 60,70, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 99.9% of allunwanted analytes (e.g., red blood-platelet enriched cells) from asample.

For example, in some embodiments the analytes of interest are retainedin an enriched solution that is less than 50, 40, 30, 20, 10, 9.0, 8.0,7.0, 6.0, 5.0, 4.5, 4.0, 3.5, 3.0, 2.5, 2.0, 1.5, 1.0, or 0.5 folddiluted from the original sample. In some embodiments, any or all of theenrichment steps increase the concentration of the analyte of interest(fetal cell), for example, by transferring them from the fluid sample toan enriched fluid sample (sometimes in a new fluid medium, such as abuffer).

III. Sample Analysis

In some embodiments, the methods herein are used for detecting thepresence or conditions of rare cells that are in a mixed sample(optionally even after enrichment) at a concentration of up to 90%, 80%,70%, 60%, 50%, 40%, 30%, 20%, 10%, 5% or 1% of all cells in the mixedsample, or at a concentration of less than 1:2, 1:4, 1:10, 1:50, 1:100,1:200, 1:500, 1:1000, 1:2000, 1:5000, 1:10,000, 1:20,000, 1:50,000,1:100,000, 1:200,000, 1:1,000,000, 1:2,000,000, 1:5,000,000,1:10,000,000, 1:20,000,000, 1:50,000,000 or 1:100,000,000 of all cellsin the sample, or at a concentration of less than 1×10⁻³, 1×10⁻⁴,1×10⁻⁵, 1×10⁻⁶, or 1×10⁻⁷ cells/4 of a fluid sample. In someembodiments, the mixed sample has a total of up to 2, 3, 4, 5, 6, 7, 8,9, 10, 15, 20, 30, 40, 50, or 100 rare cells.

The rare cells can be, for example, fetal cells derived from a maternalsample (e.g., blood sample), or epithelial, endothelial, CTC's or othercells derived from an animal to be diagnosed.

Enriched target cells (e.g., fnRBC) can be “binned” prior to analysis ofthe enriched cells (FIGS. 31 and 32). Binning is any process whichresults in the reduction of complexity and/or total cell number of theenriched cell output. Binning may be performed by any method known inthe art or described herein. One method of binning the enriched cells isby serial dilution. Such dilution may be carried out using anyappropriate platform (e.g., PCR wells, microtiter plates). Other methodsinclude nanofluidic systems which separate samples into droplets (e.g.,BioTrove, Raindance, Fluidigm). Such nanofluidic systems may result inthe presence of a single cell present in a nanodroplet.

Binning may be preceded by positive selection for target cellsincluding, but not limited to affinity binding (e.g. using anti-CD71antibodies). Alternately, negative selection of non-target cells mayprecede binning. For example, output from the size-based separationmodule may be passed through a magnetic hemoglobin enrichment module(MHEM) which selectively removes WBCs from the enriched sample.

For example, the possible cellular content of output from enrichedmaternal blood which has been passed through a size-based separationmodule (with or without further enrichment by passing the enrichedsample through a MHEM) may consist of: 1) approximately 20 fnRBC; 2)1,500 mnRBC; 3) 4,000-40,000 WBC; 4) 15×10⁶ RBC. If this sample isseparated into 100 bins (PCR wells or other acceptable binningplatform), each bin would be expected to contain: 1) 80 negative binsand 20 bins positive for one fnRBC; 2) 150 mnRBC; 3) 400-4,000 WBC; 4)15×10⁴ RBC. If separated into 10,000 bins, each bin would be expected tocontain: 1) 9,980 negative bins and 20 bins positive for one fnRBC; 2)8,500 negative bins and 1,500 bins positive for one mnRBC; 3)<1-4 WBC;4) 15×10² RBC. One of skill in the art will recognize that the number ofbins may be increased depending on experimental design and/or theplatform used for binning. The reduced complexity of the binned cellpopulations may facilitate further genetic and cellular analysis of thetarget cells.

Analysis may be performed on individual bins to confirm the presence oftarget cells (e.g. fnRBC) in the individual bin. Such analysis mayconsist of any method known in the art, including, but not limited to,FISH, PCR, STR detection, SNP analysis, biomarker detection, andsequence analysis (FIGS. 31 and 32).

IV. Fetal Biomarkers

In some embodiments fetal biomarkers may be used to detect and/orisolate fetal cells, after enrichment or after detection of fetalabnormality or lack thereof. For example, this may be performed bydistinguishing between fetal and maternal nRBCs based on relativeexpression of a gene (e.g., DYS1, DYZ, CD-71, ε- and ζ-globin) that isdifferentially expressed during fetal development. In preferredembodiments, biomarker genes are differentially expressed in the firstand/or second trimester. “Differentially expressed,” as applied tonucleotide sequences or polypeptide sequences in a cell or cell nuclei,refers to differences in over/under-expression of that sequence whencompared to the level of expression of the same sequence in anothersample, a control or a reference sample. In some embodiments, expressiondifferences can be temporal and/or cell-specific. For example, forcell-specific expression of biomarkers, differential expression of oneor more biomarkers in the cell(s) of interest can be higher or lowerrelative to background cell populations. Detection of such difference inexpression of the biomarker may indicate the presence of a rare cell(e.g., fnRBC) versus other cells in a mixed sample (e.g., backgroundcell populations). In other embodiments, a ratio of two or more suchbiomarkers that are differentially expressed can be measured and used todetect rare cells.

In one embodiment, fetal biomarkers comprise differentially expressedhemoglobins. Erythroblasts (nRBCs) are very abundant in the early fetalcirculation, virtually absent in normal adult blood and by having ashort finite lifespan, there is no risk of obtaining fnRBC which maypersist from a previous pregnancy. Furthermore, unlike trophoblastcells, fetal erythroblasts are not prone to mosaic characteristics.

Yolk sac erythroblasts synthesize ε-, ζ-, γ- and α-globins, thesecombine to form the embryonic hemoglobins. Between six and eight weeks,the primary site of erythropoiesis shifts from the yolk sac to theliver, the three embryonic hemoglobins are replaced by fetal hemoglobin(HbF) as the predominant oxygen transport system, and ε- and ζ-globinproduction gives way to γ-, α- and β-globin production within definitiveerythrocytes (Peschle et al., 1985). HbF remains the principalhemoglobin until birth, when the second globin switch occurs andβ-globin production accelerates.

Hemoglobin (Hb) is a heterodimer composed of two identical a globinchains and two copies of a second globin. Due to differential geneexpression during fetal development, the composition of the second chainchanges from ε globin during early embryonic development (1 to 4 weeksof gestation) to γ globin during fetal development (6 to 8 weeks ofgestation) to 3 globin in neonates and adults as illustrated in (Table1).

TABLE 1 Relative expression of ε, γ and β in maternal and fetal RBCs. εγ B 1^(st) trimester Fetal ++ ++ − Maternal − +/− ++ 2^(nd) trimesterFetal − ++ +/− Maternal − +/− ++

In the late-first trimester, the earliest time that fetal cells may besampled by CVS, fnRBCs contain, in addition to α globin, primarily ε andγ globin. In the early to mid second trimester, when amniocentesis istypically performed, fnRBCs contain primarily γ globin with some adult βglobin. Maternal cells contain almost exclusively α and β globin, withtraces of γ detectable in some samples. Therefore, by measuring therelative expression of the ε, γ and β genes in RBCs purified frommaternal blood samples, the presence of fetal cells in the sample can bedetermined. Furthermore, positive controls can be utilized to assessfailure of the FISH analysis itself.

In various embodiments, fetal cells are distinguished from maternalcells based on the differential expression of hemoglobins β, γ or ε.Expression levels or RNA levels can be determined in the cytoplasm or inthe nucleus of cells. Thus in some embodiments, the methods hereininvolve determining levels of messenger RNA (mRNA), ribosomal RNA(rRNA), or nuclear RNA (nRNA).

In some embodiments, identification of fnRBCs can be achieved bymeasuring the levels of at least two hemoglobins in the cytoplasm ornucleus of a cell. In various embodiments, identification and assay isfrom 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 or 20 fetal nuclei. Furthermore,total nuclei arrayed on one or more slides can number from about 100,200, 300, 400, 500, 700, 800, 5000, 10,000, 100,000, 1,000,000,2,000,000 to about 3,000,000. In some embodiments, a ratio for γ/β orε/β is used to determine the presence of fetal cells, where a numberless than one indicates that a fnRBC(s) is not present. In someembodiments, the relative expression of γ/β or ε/β provides a fnRBCindex (“FNI”), as measured by γ or ε relative to β. In some embodiments,a FNI for γ/β greater than 5, 10, 15, 20, 25, 30, 35, 40, 45, 90, 180,360, 720, 975, 1020, 1024, 1250 to about 1250, indicate that a fnRBC(s)is present. In yet other embodiments, a FNI for γ/β of less than about 1indicates that a fnRBC(s) is not present. Preferably, the above FNI isdetermined from a sample obtained during a first trimester. However,similar ratios can be used during second trimester and third trimester.

In some embodiments, the expression levels are determined by measuringnuclear RNA transcripts including, nascent or unprocessed transcripts.In another embodiment, expression levels are determined by measuringmRNA, including ribosomal RNA. There are many methods known in the artfor imaging (e.g., measuring) nucleic acids or RNA including, but notlimited to, using expression arrays from Affymetrix, Inc. or Illumina,Inc.

RT-PCR primers can be designed by targeting the globin variable regions,selecting the amplicon size, and adjusting the primers annealingtemperature to achieve equal PCR amplification efficiency. Thus TaqManprobes can be designed for each of the amplicons with well-separatedfluorescent dyes, Alexa fluor®-355 for ε, Alexa Fluor®-488 for γ, andAlexa Fluor-555 for β. The specificity of these primers can be firstverified using ε, γ, and β cDNA as templates. The primer sets that givethe best specificity can be selected for further assay development. Asan alternative, the primers can be selected from two exons spanning anintron sequence to amplify only the mRNA to eliminate the genomic DNAcontamination.

The primers selected can be tested first in a duplex format to verifytheir specificity, limit of detection, and amplification efficiencyusing target cDNA templates. The best combinations of primers can befurther tested in a triplex format for its amplification efficiency,detection dynamic range, and limit of detection.

Various commercially available reagents are available for RT-PCR, suchas One-step RT-PCR reagents, including Qiagen One-Step RT-PCR Kit andApplied Biosytems TaqMan One-Step RT-PCR Master Mix Reagents kit. Suchreagents can be used to establish the expression ratio of ε, γ, and βusing purified RNA from enriched samples. Forward primers can be labeledfor each of the targets, using Alexa fluor-355 for ε, Alexa fluor-488for γ, and Alexa fluor-555 for β. Enriched cells can be deposited bycytospinning onto glass slides. Additionally, cytospinning the enrichedcells can be performed after in situ RT-PCR. Thereafter, the presence ofthe fluorescent-labeled amplicons can be visualized by fluorescencemicroscopy. The reverse transcription time and PCR cycles can beoptimized to maximize the amplicon signal:background ratio to havemaximal separation of fetal over maternal signature. Preferably,signal:background ratio is greater than 5, 10, 50 or 100 and the overallcell loss during the process is less than 50, 10 or 5%.

V. Fetal Cell Analysis

Fetal conditions that can be determined based on the methods and systemsherein include the presence of a fetus and/or a condition of the fetussuch as fetal aneuploidy e.g., trisomy 13, trisomy 18, trisomy 21 (DownSyndrome), Klinefelter Syndrome (XXY) and other irregular number of sexor autosomal chromosomes, including monosomy of one or more chromosomes(X chromosome monosomy, also known as Turner's syndrome), trisomy of oneor more chromosomes (13, 18, 21, and X), tetrasomy and pentasomy of oneor more chromosomes (which in humans is most commonly observed in thesex chromosomes, e.g. XXXX, XXYY, XXXY, XYYY, XXXXX, XXXXY, XXXYY, XYYYYand XXYYY), monoploidy, triploidy (three of every chromosome, e.g. 69chromosomes in humans), tetraploidy (four of every chromosome, e.g. 92chromosomes in humans), pentaploidy and multiploidy. Other fetalconditions that can be detected using the methods herein includesegmental aneuploidy, such as 1p36 duplication, dup(17)(p11.2p11.2)syndrome, Down syndrome, Pre-eclampsia. Pre-term labor, Edometriosis,Pelizaeus-Merzbacher disease, dup(22)(q11.2q11.2) syndrome, Cat eyesyndrome. In some embodiment, the fetal abnormality to be detected isdue to one or more deletions in sex or autosomal chromosomes, includingCri-du-chat syndrome, Wolf-Hirschhorn syndrome, Williams-Beurensyndrome, Charcot-Marie-Tooth disease, Hereditary neuropathy withliability to pressure palsies, Smith-Magenis syndrome,Neurofibromatosis, Alagille syndrome, Velocardiofacial syndrome,DiGeorge syndrome, steroid sulfatase deficiency, Kallmann syndrome,Microphthalmia with linear skin defects, Adrenal hypoplasia, Glycerolkinase deficiency, Pelizaeus-Merzbacher disease, testis-determiningfactor on Y, Azospermia (factor a), Azospermia (factor b), Azospermia(factor c) and 1p36 deletion. In some cases, the fetal abnormality is anabnormal decrease in chromosomal number, such as XO syndrome.

Conditions in a patient that can be detected using the systems andmethods herein include, infection (e.g., bacterial, viral, or fungalinfection), neoplastic or cancer conditions (e.g., acute lymphoblasticleukemia, acute or chronic lymphocyctic or granulocytic tumor, acutemyeloid leukemia, acute promyelocytic leukemia, adenocarcinoma, adenoma,adrenal cancer, basal cell carcinoma, bone cancer, brain cancer, breastcancer, bronchi cancer, cervical dysplasia, chronic myelogenousleukemia, colon cancer, epidermoid carcinoma, Ewing's sarcoma,gallbladder cancer, gallstone tumor, giant cell tumor, glioblastomamultiforma, hairy-cell tumor, head cancer, hyperplasia, hyperplasticcorneal nerve tumor, in situ carcinoma, intestinal ganglioneuroma, isletcell tumor, Kaposi's sarcoma, kidney cancer, larynx cancer, leiomyomatertumor, liver cancer, lung cancer, lymphomas, malignant carcinoid,malignant hypercalcemia, malignant melanomas, marfanoid habitus tumor,medullary carcinoma, metastatic skin carcinoma, mucosal neuromas,mycosis fungoide, myelodysplastic syndrome, myeloma, neck cancer, neuraltissue cancer, neuroblastoma, osteogenic sarcoma, osteosarcoma, ovariantumor, pancreas cancer, parathyroid cancer, pheochromocytoma,polycythemia Vera, primary brain tumor, prostate cancer, rectum cancer,renal cell tumor, retinoblastoma, rhabdomyosarcoma, seminoma, skincancer, small-cell lung tumor, soft tissue sarcoma, squamous cellcarcinoma, stomach cancer, thyroid cancer, topical skin lesion,veticulum cell sarcoma, or Wilm's tumor), inflammation, etc.

In some cases, sample analyses involves performing one or more geneticanalyses or detection steps on nucleic acids from the enriched product(e.g., enriched cells or nuclei). Nucleic acids from enriched cells orenriched nuclei that can be analyzed by the methods herein include:double-stranded DNA, single-stranded DNA, single-stranded DNA hairpins,DNA/RNA hybrids, RNA (e.g. mRNA) and RNA hairpins. Examples of geneticanalyses that can be performed on enriched cells or nucleic acidsinclude, e.g., SNP detection, STR detection, and RNA expressionanalysis.

In some embodiments, less than 1 μg, 500 ng, 200 ng, 100 ng, 50 ng, 40ng, 30 ng, 20 ng, 10 ng, 5 ng, 1 ng, 500 pg, 200 pg, 100 pg, 50 pg, 40pg, 30 pg, 20 pg, 10 pg, 5 pg, or 1 pg of nucleic acids are obtainedfrom the enriched sample for further genetic analysis. In some cases,about 1-5 μg, 5-10 μg, or 10-100 μg of nucleic acids are obtained fromthe enriched sample for further genetic analysis.

When analyzing, for example, a sample such as a blood sample from apatient to diagnose a condition such as cancer, the genetic analyses canbe performed on one or more genes encoding or regulating a polypeptidelisted in FIG. 5. In some cases, a diagnosis is made by comparingresults from such genetic analyses with results from similar analysesfrom a reference sample (one without fetal cells or CTC's, as the casemay be). For example, a maternal blood sample enriched for fetal cellscan be analyzed to determine the presence of fetal cells and/or acondition in such cells by comparing the ratio of maternal to paternalgenomic DNA (or alleles) in control and test samples.

In some embodiments, target nucleic acids from a test sample areamplified and optionally results are compared with amplification ofsimilar target nucleic acids from a non-rare cell population (referencesample). Amplification of target nucleic acids can be performed by anymeans known in the art. In some cases, target nucleic acids areamplified by polymerase chain reaction (PCR). Examples of PCR techniquesthat can be used include, but are not limited to, quantitative PCR,quantitative fluorescent PCR (QF-PCR), multiplex fluorescent PCR(MF-PCR), real time PCR(RT-PCR), single cell. PCR, restriction fragmentlength polymorphism PCR(PCR-RFLP), PCR-RFLP/RT-PCR-RFLP, hot start PCR,nested PCR, in situ polonony PCR, in situ rolling circle amplification(RCA), bridge PCR, picotiter PCR and emulsion PCR. Other suitableamplification methods include the ligase chain reaction (LCR),transcription amplification, self-sustained sequence replication,selective amplification of target polynucleotide sequences, consensussequence primed polymerase chain reaction (CP-PCR), arbitrarily primedpolymerase chain reaction (AP-PCR), degenerate oligonucleotide-primedPCR (DOP-PCR) and nucleic acid based sequence amplification (NABSA).Other amplification methods that can be used herein include thosedescribed in U.S. Pat. Nos. 5,242,794; 5,494,810; 4,988,617; and6,582,938.

In any of the embodiments, amplification of target nucleic acids mayoccur on a bead. In any of the embodiments herein, target nucleic acidsmay be obtained from a single cell.

In any of the embodiments herein, the nucleic acid(s) of interest can bepre-amplified prior to the amplification step (e.g., PCR). In somecases, a nucleic acid sample may be pre-amplified to increase theoverall abundance of genetic material to be analyzed (e.g., DNA).Pre-amplification can therefore include whole genome amplification suchas multiple displacement amplification (MDA) or amplifications withouter primers in a nested PCR approach.

In some embodiments amplified nucleic acid(s) are quantified. Methodsfor quantifying nucleic acids are known in the art and include, but arenot limited to, gas chromatography, supercritical fluid chromatography,liquid chromatography (including partition chromatography, adsorptionchromatography, ion exchange chromatography, size-exclusionchromatography, thin-layer chromatography, and affinity chromatography),electrophoresis (including capillary electrophoresis, capillary zoneelectrophoresis, capillary isoelectric focusing, capillaryelectrochromatography, micellar electrokinetic capillary chromatography,isotachophoresis, transient isotachophoresis and capillary gelelectrophoresis), comparative genomic hybridization (CGH), microarrays,bead arrays, and high-throughput genotyping such as with the use ofmolecular inversion probe (MIP).

Quantification of amplified target nucleic acid can be used to determinegene/or allele copy number, gene or exon-level expression,methylation-state analysis, or detect a novel transcript in order todiagnose or condition, i.e. fetal abnormality or cancer.

In some embodiments, analysis involves detecting one or more mutationsor SNPs in DNA from e.g., enriched rare cells or enriched rare DNA. Suchdetection can be performed using, for example, DNA microarrays. Examplesof DNA microarrays include those commercially available from Affymetrix,Inc. (Santa Clara, Calif.), including the GeneChip™ Mapping Arraysincluding Mapping 100K Set, Mapping 10K 2.0 Array, Mapping 10K Array,Mapping 500K Array Set, and GeneChip™ Human Mitochondrial ResequencingArray 2.0. The Mapping 10K array, Mapping 100K array set, and Mapping500K array set analyze more than 10,000, 100,000 and 500,000 differenthuman SNPs, respectively. SNP detection and analysis using GeneChip™Mapping Arrays is described in part in Kennedy, G. C., et al., NatureBiotechnology 21, 1233-1237, 2003; Liu, W. M., Bioinformatics 19,2397-2403, 2003; Matsuzaki, H., Genome Research 3, 414-25, 2004; andMatsuzaki, H., Nature Methods, 1, 109-111, 2004 as well as in U.S. Pat.Nos. 5,445,934; 5,744,305; 6,261,776; 6,291,183; 5,799,637; 5,945,334;6,346,413; 6,399,365; and 6,610,482, and EP 619 321; 373 203. In someembodiments, a microarray is used to detect at least 5, 10, 20, 50, 100,200, 500, 1,000, 2,000, 5,000 10,000, 20,000, 50,000, 100,000, 200,000,or 500,000 different nucleic acid target(s) (e.g., SNPs, mutations orSTRs) in a sample.

Methods for analyzing chromosomal copy number using mapping arrays aredisclosed, for example, in Bignell et al., Genome Res. 14:287-95 (2004),Lieberfarb, et al., Cancer Res. 63:4781-4785 (2003), Zhao et al., CancerRes. 64:3060-71 (2004), Nannya et al., Cancer Res. 65:6071-6079 (2005)and Ishikawa et al., Biochem. and Biophys. Res. Comm., 333:1309-1.314(2005). Computer implemented methods for estimation of copy number basedon hybridization intensity are disclosed in U.S. Publication ApplicationNos. 20040157243; 20050064476; and 20050130217.

In preferred aspects, mapping analysis using fixed content arrays, forexample, 10K, 100K or 500K arrays, preferably identify one or a fewregions that show linkage or association with the phenotype of interest.Those linked regions may then be more closely analyzed to identify andgenotype polymorphisms within the identified region or regions, forexample, by designing a panel of MIPs targeting polymorphisms ormutations in the identified region. The targeted regions may beamplified by hybridization of a target specific primer and extension ofthe primer by a highly processive strand displacing polymerase, such asphi29 and then analyzed, for example, by genotyping.

A quick overview for the process of using a SNP detection microarray(such as the Mapping 100K Set) is illustrated in FIG. 6. First, in step600 a sample comprising one or more rare cells (e.g., fetal or CTC) andnon-rare cells (e.g., RBC's) is obtained from an animal such as a human.In step 601, rare cells or rare DNA (e.g., rare nuclei) are enrichedusing one or more methods disclosed herein or known in the art.Preferably, rare cells are enriched by flowing the sample through anarray of obstacles that selectively directs particles or cells ofdifferent hydrodynamic sizes into different outlets. In some cases, gDNAis obtained from both rare and non-rare cells enriched by the methodsherein.

In step 602, genomic DNA is obtained from the rare cell(s) or nuclei andoptionally one or more non-rare cells remaining in the enriched mixture.In step 603, the genomic DNA obtained from the enriched sample isdigested with a restriction enzyme, such as XbaI or Hind III. Other DNAmicroarrays may be designed for use with other restriction enzymes,e.g., Sty I or NspI. In step 604 all fragments resulting from thedigestion are ligated on both ends with an adapter sequence thatrecognizes the overhangs from the restriction digest. In step 605, theDNA fragments are diluted. Subsequently, in step 606 fragments havingthe adapter sequence at both ends are amplified using a generic primerthat recognizes the adapter sequence. The PCR conditions used foramplification preferentially amplify fragments that have a uniquelength, e.g., between 250 and 2,000 base pairs in length. In steps 607,amplified DNA sequences are fragmented, labeled and hybridized with theDNA microarray (e.g., 100K Set Array or other array). Hybridization isfollowed by a step 608 of washing and staining.

In step 609 results are visualized using a scanner that enables theviewing of intensity of data collected and a software “calls” the basespresent at each of the SNP positions analyzed. Computer implementedmethods for determining genotype using data from mapping arrays aredisclosed, for example, in Liu, et al., Bioinformatics 19:2397-2403,2003; and Di et al., Bioinformatics 21:1958-63, 2005. Computerimplemented methods for linkage analysis using mapping array data aredisclosed, for example, in Ruschendorf and Nurnberg, Bioinformatics21:2123-5, 2005; and Leykin et al., BMC Genet. 6:7, 2005; and in U.S.Pat. No. 5,733,729.

In some cases, genotyping microarrays that are used to detect SNPs canbe used in combination with molecular inversion probes (MIPs) asdescribed in Hardenbol et al., Genome Res. 15(2):269-275, 2005,Hardenbol, P. et al. Nature Biotechnology 21(6), 673-8, 2003; Faham M,et al. Hum Mol. Genet. August 1; 10(16):1657-64, 2001; Maneesh Jain,Ph.D., et all. Genetic Engineering News V24: No. 18, 2004; andFakhrai-Rad H, et al. Genome Res. July; 14(7):1404-12, 2004; and in U.S.Pat. No. 6,858,412. Universal tag arrays and reagent kits for performingsuch locus specific genotyping using panels of custom MIPs are availablefrom Affymetrix and ParAllele. MIP technology involves the useenzymological reactions that can score up to 10,000; 20,000, 50,000;100,000; 200,000; 500,000; 1,000,000; 2,000,000 or 5,000,000 SNPs(target nucleic acids) in a single assay. The enzymological reactionsare insensitive to cross-reactivity among multiple probe molecules andthere is no need for pre-amplification prior to hybridization of theprobe with the genomic DNA. In any of the embodiments, the targetnucleic acid(s) or SNPs are obtained from a single cell.

Thus, the present invention contemplates obtaining a sample enriched forfetal cells, epithelial cells or CTC's and analyzing such enrichedsample using the MIP technology or oligonucleotide probes that areprecircle probes i.e., probes that form a substantially complete circlewhen they hybridize to a SNP. The precircle probes comprise a firsttargeting domain that hybridizes upstream to a SNP position, a secondtargeting domain that hybridizes downstream of a SNP position, at leasta first universal priming site, and a cleavage site. Once the probes areallowed to contact genomic DNA regions of interest (comprising SNPs tobe assayed), a hybridization complex forms with a precircle probe and agap at a SNP position region. Subsequently, ligase is used to “fill in”the gap or complete the circle. The enzymatic “gap fill” process occursin an allele-specific manner. The nucleotide added to the probe to fillthe gap is complementary to the nucleotide base at the SNP position.Once the probe is circular, it may be separated from cross-reacted orunreacted probes by a simple exonuelease reaction. The circular probe isthen cleaved at the cleavage site such that it becomes linear again. Thecleavage site can be any site in the probe other than the SNP site.Linearization of the circular probe results in the placement ofuniversal primer region at one end of the probe. The universal primerregion can be coupled to a tag region. The tag can be detected usingamplification techniques known in the art. The SNP analyzed cansubsequently be detected by amplifying the cleaved (linearized) probe todetect the presence of the target sequence in said sample or thepresence of the tag.

Another method contemplated by the present invention to detect SNPsinvolves the use of bead arrays (e.g., such as one commerciallyavailable by Illumina, Inc.) as described in U.S. Pat. Nos. 7,040,959;7,035,740; 7,033,754; 7,025,935, 6,998,274; 6,942,968; 6,913,884;6,890,764; 6,890,741; 6,858,394; 6,846,460; 6,812,005; 6,770,441;6,663,832; 6,620,584; 6,544,732; 6,429,027; 6,396,995; 6,355,431 and USPublication Application Nos. 20060019258; 20050266432; 20050244870;20050216207; 20050181394; 20050164246; 20040224353; 20040185482;20030198573; 20030175773; 20030003490; 20020187515; and 20020177141; aswell as Shen, R., et al. Mutation Research 573 70-82 (2005).

FIG. 7 illustrates an overview of one embodiment of detecting mutationsor SNPs using bead arrays. In this embodiment, a sample comprising oneor more rare cells (e.g., fetal or CTC) and non-rare cells (e.g., RBC's)is obtained from an animal such as a human. Rare cells or rare DNA(e.g., rare nuclei) are enriched using one or more methods disclosedherein or known in the art. Preferably, rare cells are enriched byflowing the sample through an array of obstacles that selectivelydirects particles or cells of different hydrodynamic sizes intodifferent outlets.

In step 701, genomic DNA is obtained from the rare cell(s) or nucleiand, optionally, from the one or more non-rare cells remaining in theenriched mixture. The assays in this embodiment require very littlegenomic DNA starting material, e.g., between 250 ng-2 μg. Depending onthe multiplex level, the activation step may require only 160 pg of DNAper SNP geneotype call. In step 702, the genomic DNA is activated suchthat it may bind paramagnetic particles. In step 703 assayoligonucleotides, hybridization buffer, and paramagnetic particles arecombined with the activated DNA and allowed to hybridize (hybridizationstep). In some cases, three oligonucleotides are added for each SNP tobe detected. Two of the three oligos are specific for each of the twoalleles at a SNP position and are referred to as Allele-Specific Oligos(ASOs). A third oligo hybridizes several bases downstream from the SNPsite and is referred to as the Locus-Specific Oligo (LSO). All threeoligos contain regions of genomic complementarity (C1, C2, and C3) anduniversal PCR primer sites (P1, P2 and P3). The LSO also contains aunique address sequence (Address) that targets a particular bead type.(Up to 1,536 SNPs may be assayed in this manner using GoldenGate™ Assayavailable by Illumina, Inc. (San Diego, Calif.).) During the primerhybridization process, the assay oligonucleotides hybridize to thegenomic DNA sample bound to paramagnetic particles. Becausehybridization occurs prior to any amplification steps, no amplificationbias is introduced into the assay.

In step 704, following the hybridization step, several wash steps areperformed reducing noise by removing excess and mis-hybridizedoligonucleotides. Extension of the appropriate ASO and ligation of theextended product to the LSO joins information about the genotype presentat the SNP site to the address sequence on the LSO. In step 705, thejoined, full-length products provide a template for performing PCRreactions using universal PCR primers P1, P2, and P3. Universal primersP1 and P2 are labeled with two different labels (e.g., Cy3 and Cy5).Other labels that can be used include, chromophores, fluorescentmoieties, enzymes, antigens, heavy metal, magnetic probes, dyes,phosphorescent groups, radioactive materials, chemiluminescent moieties,scattering or fluorescent nanoparticles, Raman signal generatingmoieties, or electrochemical detection moieties.

In step 706, the single-stranded, labeled DNAs are eluted and preparedfor hybridization. In step 707, the single-stranded, labeled DNAs arehybridized to their complement bead type through their unique addresssequence. Hybridization of the GoldenGate Assay™ products onto the ArrayMatrix™ of Beadchip™ allows for separation of the assay products insolution, onto a solid surface for individual SNP genotype readout.

In step 708, the array is washed and dried. In step 709, a reader suchas the BeadArray Reader™ is used to analyze signals from the label. Forexample, when the labels are dye labels such as Cy3 and Cy5, the readercan analyze the fluorescence signal on the Sentrix Array Matrix orBeadChip.

In step 710, a computer program comprising a computer readable mediumhaving a computer executable logic is used to automate genotypingclusters and callings.

In any of the embodiments herein, preferably, more than 1000, 5,000,10,000, 50,000, 100,000, 500,000, or 1,000,000 SNPs are assayed inparallel.

In some embodiments, analysis involves detecting levels of expression ofone or more genes or exons in e.g., enriched rare cells or enriched raremRNA. Such detection can be performed using, for example, expressionmicroarrays. Thus, the present invention contemplates a methodcomprising the steps of: enriching rare cells from a sample as describedherein, isolating nucleic acids from the rare cells, contacting amicroarray under conditions such that the nucleic acids specificallyhybridize to the genetic probes on the microarray, and determining thebinding specificity (and amount of binding) of the nucleic acid from theenriched sample to the probes. The results from these steps can be usedto obtain a binding pattern that would reflect the nucleic acidabundance and establish a gene expression profile. In some embodiments,the gene expression or copy number results from the enriched cellpopulation is compared with gene expression or copy number of a non-rarecell population to diagnose a disease or a condition.

Examples of expression microarrays include those commercially availablefrom Affymetrix, Inc. (Santa Clara, Calif.), such as the exon arrays(e.g., Human Exon ST Array); tiling arrays (e.g., Chromosome 21/22 1.0Array Set, ENCODE01 1.0 Array, or Human Genome Arrays +); and 3′eukaryotic gene expression arrays (e.g., Human Genome Array +, etc.).Examples of human genome arrays include HuGene FL Genome Array, HumanCancer G110 ARray, Human Exon 1.0 ST, Human Genome Focus Array, HumanGenome U133 Plus 2.0, Human Genome U133 Set, Human Genome U133A 2.0,Human Promoter U95 SetX, Human Tiling 1.0R Array Set, Human Tiling 2.0RArray Set, and Human X3P Array.

Expression detection and analysis using microarrays is described in partin Valk, P. J. et al. New England Journal of Medicine 350(16), 1617-28,2004; Modlich, O. et al. Clinical Cancer Research 10(10), 3410-21, 2004;Onken, Michael D. et al. Cancer Res. 64(20), 7205-7209, 2004; Gardian,et al. J. Biol. Chem. 280(1), 556-563, 2005; Becker, M. et al. Mol.Cancer. Ther. 4(1), 151-170, 2005; and Flechner, S M et al. Am JTransplant 4(9), 1475-89, 2004; as well as in U.S. Pat. Nos. 5,445,934;5,700,637; 5,744,305; 5,945,334; 6,054,270; 6,140,044; 6,261,776;6,291,183; 6,346,413; 6,399,365; 6,420,169; 6,551,817; 6,610,482;6,733,977; and EP 619 321; 323 203.

An overview of a protocol that can be used to detect RNA expression(e.g., using Human Genome U133A Set) is illustrated in FIG. 8. In step800 a sample comprising one or more rare cells (e.g., fetal, epithelialor CTC) and non-rare cells (e.g., RBC's) is obtained from an animal,such as a human. In step 801, rare cells or rare DNA (e.g., rare nuclei)are enriched using one or more methods disclosed herein or known in theart. Preferably, rare cells are enriched by flowing the sample throughan array of obstacles that selectively directs particles or cells ofdifferent hydrodynamic sizes into different outlets such that rare cellsand cells larger than rare cells are directed into a first outlet andone or more cells or particles smaller than the rare cells are directedinto a second outlet.

In step 802 total RNA or poly-A mRNA is obtained from enriched cell(s)(e.g., fetal, epithelial or CTC's) using purification techniques knownin the art. Generally, about 1 μg-2 μg of total RNA is sufficient. Instep 803, a first-strand complementary DNA (cDNA) is synthesized usingreverse transcriptase and a single T7-oligo(dT) primer. In step 804, asecond-strand cDNA is synthesized using DNA ligase, DNA polymerase, andRNase enzyme. In step 805, the double stranded cDNA (ds-cDNA) ispurified. In step 806, the ds-cDNA serves as a template for in vitrotranscription reaction. The in vitro transcription reaction is carriedout in the presence of T7 RNA polymerase and a biotinylated nucleotideanalog/ribonucleotide mix. This generates roughly ten times as manycomplementary RNA (cRNA) transcripts.

In step 807, biotinylated cRNAs are cleaned up, and subsequently in step808, they are fragmented randomly. Finally, in step 809 the expressionmicroarray (e.g., Human Genome U133 Set) is washed with the fragmented,biotin-labeled cRNAs and subsequently stained with streptavidinphycoerythrin (SAPE). And in step 810, after final washing, themicrarray is scanned to detect hybridization of cRNA to probe pairs.

In step 811 a computer program product comprising a computer executablelogic analyzes images generated from the scanner to determine geneexpression. Such methods are disclosed in part in U.S. Pat. No.6,505,125.

Another method contemplated by the present invention to detect andquantify gene expression involves the use of bead as is commerciallyavailable by Illumina, Inc. (San Diego) and as described in U.S. Pat.Nos. 7,035,740; 7,033,754; 7,025,935, 6,998,274; 6,942,968; 6,913,884;6,890,764; 6,890,741; 6,858,394; 6,812,005; 6,770,441; 6,620,584;6,544,732; 6,429,027; 6,396,995; 6,355,431 and US PublicationApplication Nos. 20060019258; 20050266432; 20050244870; 20050216207;20050181394; 20050164246; 20040224353; 20040185482; 20030198573;20030175773; 20030003490; 20020187515; and 20020177141; and in B. E.Stranger, et al., Public Library of Science-Genetics, 1 (6), December2005; Jingli Cai, et al., Stem Cells, published online Nov. 17, 2005; C.M. Schwartz, et al., Stem Cells and Development, 14, 517-534, 2005;Barnes, M., J. et al., Nucleic Acids Research, 33 (18), 5914-5923,October 2005; and Bibikova M, et al. Clinical. Chemistry, Volume 50, No.12, 2384-2386, December 2004.

FIG. 9 illustrates an overview of one embodiment of detecting mutationsor SNPs using bead arrays. In step 900 a sample comprising one or morerare cells (e.g., fetal, epithelial or CTC) and non-rare cells (e.g.,RBC's) is obtained from an animal, such as a human. In step 901, rarecells or rare DNA (e.g., rare nuclei) are enriched using one or moremethods disclosed herein or known in the art. Preferably, rare cells areenriched by flowing the sample through an array of obstacles thatselectively directs particles or cells of different hydrodynamic sizesinto different outlets such that rare cells and cells larger than rarecells are directed into a first outlet and one or more cells orparticles smaller than the rare cells are directed into a second outlet.

In step 902, total RNA is extracted from enriched cells (e.g., fetalcells, CTC, or epithelial cells). In step 903, two one-quarter scaleMessage Amp II reactions (Ambion, Austin, Tex.) are performed for eachRNA extraction using 200 ng of total RNA. MessageAmp is a procedurebased on antisense RNA (aRNA) amplification, and involves a series ofenzymatic reactions resulting in linear amplification of exceedinglysmall amounts of RNA for use in array analysis. Unlike exponential RNAamplification methods, such as NASBA and RT-PCR, aRNA amplificationmaintains representation of the starting mRNA population. The procedurebegins with total or poly(A) RNA that is reverse transcribed using aprimer containing both oligo(dT) and a T7 RNA polymerase promotersequence. After first-strand synthesis, the reaction is treated withRNase H to cleave the mRNA into small fragments. These small RNAfragments serve as primers during a second-strand synthesis reactionthat produces a double-stranded cDNA template for transcription.Contaminating rRNA, mRNA fragments and primers are removed and the cDNAtemplate is then used in a large scale in vitro transcription reactionto produce linearly amplified aRNA. The aRNA can be labeled with biotinrNTPS or amino allyl-UTP during transcription.

In step 904, biotin-16-UTP (Perkin Elmer, Wellesley, Calif.) is addedsuch that half of the UTP is used in the in vitro transcriptionreaction. In step 905, cRNA yields are quantified using RiboGreen(Invitrogen, Carlsbad, Calif.). In step 906, 1 μg of cRNA is hybridizedto a bead array (e.g., Illumina Bead Array). In step 907, one or morewashing steps is performed on the array. In step 908, after finalwashing, the micrarray is scanned to detect hybridization of cRNA. Instep 908, a computer program product comprising an executable programanalyzes images generated from the scanner to determine gene expression.

Additional description for preparing RNA for bead arrays is described inKacharmina J E, et al., Methods Enzymol 303: 3-18, 1999; Pabon C, etal., Biotechniques 31(4): 874-9, 2001; Van Gelder R N, et al., Proc NatlAcad Sci USA 87: 1663-7 (1990); and Murray, S S. BMC. Genetics 6(Suppl1):S85 (2005).

Preferably, more than 1000, 5,000, 10,000, 50,000, 100,000, 500,000, or1,000,000 transcripts are assayed in parallel.

In any of the embodiments herein, genotyping (e.g., SNP detection)and/or expression analysis (e.g., RNA transcript quantification) ofgenetic content from enriched rare cells or enriched rare cell nucleican be accomplished by sequencing. Sequencing can be accomplishedthrough classic Sanger sequencing methods which are well known in theart. Sequence can also be accomplished using high-throughput systemssome of which allow detection of a sequenced nucleotide immediatelyafter or upon its incorporation into a growing strand, i.e., detectionof sequence in real time or substantially real time. In some cases, highthroughput sequencing generates at least 1,000, at least 5,000, at least10,000, at least 20,000, at least 30,000, at least 40,000, at least50,000, at least 100,000 or at least 500,000 sequence reads per hour;with each read being at least 50, at least 60, at least 70, at least 80,at least 90, at least 100, at least 120 or at least 150 bases per read.Sequencing can be preformed using genomic DNA or cDNA derived from RNAtranscripts as a template.

In some embodiments, high-throughput sequencing involves the use oftechnology available by Helicos BioSciences Corporation (Cambridge,Mass.) such as the Single Molecule Sequencing by Synthesis (SMSS)method. SMSS is unique because it allows for sequencing the entire humangenome in up to 24 hours. This fast sequencing method also allows fordetection of a SNP/nucleotide in a sequence in substantially real timeor real time. Finally, SMSS is powerful because, like the MIPtechnology, it does not require a preamplification step prior tohybridization. In fact, SMSS does not require any amplification. SMSS isdescribed in part in US Publication Application Nos. 20060024711;20060024678; 20060012793; 20060012784; and 20050100932.

An overview the use of SMSS for analysis of enriched cells/nucleic acids(e.g., fetal cells, epithelial cells, CTC's) is outlined in FIG. 10.

First, in step 1000 a sample comprising one or more rare cells (e.g.,fetal or CTC) and one or more non-rare cells (e.g., RBC's) is obtainedfrom an animal, such as a human. In step 1002, rare cells or rare DNA(e.g., rare nuclei) are enriched using one or more methods disclosedherein or known in the art. Preferably, rare cells are enriched byflowing the sample through an array of obstacles that selectivelydirects particles or cells of different hydrodynamic sizes intodifferent outlets. In step 1004, genomic DNA is obtained from the rarecell(s) or nuclei and optionally one or more non-rare cells remaining inthe enriched mixture.

In step 1006 the genomic DNA is purified and optionally fragmented. Instep 1008, a universal priming sequence is generated at the end of eachstrand. In step 1010, the strands are labeled with a fluorescentnucleotide. These strands will serve as templates in the sequencingreactions.

In step 1012 universal primers are immobilized on a substrate (e.g.,glass surface) inside a flow cell.

In step 1014, the labeled DNA strands are hybridized to the immobilizedprimers on the substrate.

In step 1016, the hybridized DNA strands are visualized by illuminatingthe surface of the substrate with a laser and imaging the labeled DNAwith a digital TV camera connected to a microscope. In this step, theposition of all hybridization duplexes on the surface is recorded.

In step 1018, DNA polymerase is flowed into the flow cell. Thepolymerase catalyzes the addition of the labeled nucleotides to thecorrect primers.

In step 1020, the polymerase and unincorporated nucleotides are washedaway in one or more washing procedures.

In step 1022, the incorporated nucleotides are visualized byilluminating the surface with a laser and imaging the incorporatednucleotides with a camera. In this step, recordation is made of thepositions of the incorporated nucleotides.

In step 1024, the fluorescent labels on each nucleotide are removed.

Steps 1018-1024 are repeated with the next nucleotide such that thesteps are repeated for A, G, T, and C. This sequence of events isrepeated until the desired read length is achieved.

SMSS can be used, e.g., to sequence DNA from enriched CTC's to identifygenetic mutations (e.g., SNPs) in DNA, or to profile gene expression ofmRNA transcrips of such cells or other cells (fetal cells). SMSS canalso be used to identify genes in CTC's that are methylated (“turnedoff”) and develop cancer diagnostics based on such methylation. Finally,enriched cells/DNA can be analyzed using SMSS to detect minute levels ofDNA from pathogens such as viruses, bacteria or fungi. Such DNA analysiscan further be used for serotyping to detect, e.g., drug resistance orsusceptibility to disease. Furthermore, enriched stem cells can beanalyzed using SMSS to determine if various expression profiles anddifferentiation pathways are turned “on” or “off”. This allows for adetermination to be made of the enriched stem cells are prior to or postdifferentiation.

In some embodiments, high-throughput sequencing involves the use oftechnology available by 454 Lifesciences, Inc. (Branford, Conn.) such asthe PicoTiterPlate device which includes a fiber optic plate thattransmits chemilluminescent signal generated by the sequencing reactionto be recorded by a CCD camera in the instrument. This use of fiberoptics allows for the detection of a minimum of 20 million base pairs in4.5 hours.

Methods for using bead amplification followed by fiber optics detectionare described in Marguiles, M., et al. “Genome sequencing inmicrofabricated high-density pricolitre reactors”, Nature,doi:10.1038/nature03959; and well as in US Publication Application Nos.20020012930; 20030068629; 20030100102; 20030148344; 20040248161;20050079510, 20050124022; and 20060078909.

An overview of this embodiment is illustrated in FIG. 11.

First, in step 1100 a sample comprising one or more rare cells (e.g.,fetal, epithelial or CTC) and one or more non-rare cells (e.g., RBC's)is obtained from an animal, such as a human. In step 1102, rare cells orrare DNA (e.g., rare nuclei) are enriched using one or more methodsdisclosed herein, or known in the art. Preferably, rare cells areenriched by flowing the sample through an array of obstacles thatselectively directs particles or cells of different hydrodynamic sizesinto different outlets. In step 1104, genomic DNA is obtained from therare cell(s) or nuclei and optionally one or more non-rare cellsremaining in the enriched mixture.

In step 1112, the enriched genomic DNA is fragmented to generate alibrary of hundreds of DNA fragments for sequencing runs. Genomic DNA(gDNA) is fractionated into smaller fragments (300-500 base pairs) thatare subsequently polished (blunted). In step 1113, short adaptors (e.g.,A and B) are ligated onto the ends of the fragments. These adaptorsprovide priming sequences for both amplification and sequencing of thesample-library fragments. One of the adaptors (e.g., Adaptor B) containsa 5′-biotin tag or other tag that enables immobilization of the libraryonto beads (e.g., streptavidin coated beads). In step 1114, only gDNAfragments that include both Adaptor A and B are selected usingavidin-blotting purification. The sstDNA library is assessed for itsquality and the optimal amount (DNA copies per bead) needed forsubsequent amplification is determined by titration. In step 1115, thesstDNA library is annealed and immobilized onto an excess of capturebeads (e.g., streptavidin coated beads). The latter occurs underconditions that favor each bead to carry only a single sstDNA molecule.In step 1116, each bead is captured in its own microreactor, such as awell, which may optionally be addressable, or a picolitre-sized well. Instep 1117, the bead-bound library is amplified using, e.g., emPCR. Thiscan be accomplished by capturing each bead within a droplet of aPCR-reaction-mixture-in-oil-emulsion. Thus, the bead-bound library canbe emulsified with the amplification reagents in a water-in-oil mixture.EmPCR enables the amplification of a DNA fragment immobilized on a beadfrom a single fragment to 10 million identical copies. Thisamplification step generates sufficient identical DNA fragments toobtain a strong signal in the subsequent sequencing step. Theamplification step results in bead-immobilized, clonally amplified. DNAfragments. The amplification on the bead results can result in each beadcarrying at least one million, at least 5 million, or at least 10million copies of the unique target nucleic acid.

The emulsion droplets can then be broken, genomic material on each beadmay be denatured, and single-stranded nucleic acids clones can bedeposited into wells, such as picolitre-sized wells, for furtheranalysis including, but are not limited to quantifying said amplifiednucleic acid, gene and exon-level expression analysis, methylation-stateanalysis, navel transcript discovery, sequencing, genotyping orresequencing. In step 1118, the sstDNA library beads are added to a DNAbead incubation mix (containing DNA polymerase) and are layered withenzyme beads (containing sulfurylase and luciferase as is described inU.S. Pat. Nos. 6,956,114 and 6,902,921) onto a fiber optic plate such asthe PicoTiterPlate device. The fiber optic plate is centrifuged todeposit the beads into wells (˜up to 50 or 45 microns in diameter). Thelayer of enzyme beads ensures that the DNA beads remain positioned inthe wells during the sequencing reaction. The bead-deposition processmaximizes the number of wells that contain a single amplified librarybead (avoiding more than one sstDNA library bead per well). Preferably,each well contains a single amplified library bead. In step 1119, theloaded fiber optic plate (e.g., PicoTiterPlate device) is then placedinto a sequencing apparatus (e.g., the Genome Sequencer 20 Instrument).Fluidics subsystems flow sequencing reagents (containing buffers andnucleotides) across the wells of the plate. Nucleotides are flowedsequentially in a fixed order across the fiber optic plate during asequencing run. In step 1120, each of the hundreds of thousands of beadswith millions of copies of DNA is sequenced in parallel during thenucleotide flow. If a nucleotide complementary to the template strand isflowed into a well, the polymerase extends the existing DNA strand byadding nucleotide(s) which transmits a chemilluminescent signal. In step1122, the addition of one (or more) nucleotide(s) results in a reactionthat generates a chemilluminescent signal that is recorded by a digitalcamera or CCD camera in the instrument. The signal strength of thechemilluminescent signal is proportional to the number of nucleotidesadded. Finally, in step 1124, a computer program product comprising anexecutable logic processes the chemilluminescent signal produced by thesequencing reaction. Such logic enables whole genome sequencing for denovo or resequencing projects.

In some embodiments, high-throughput sequencing is performed usingClonal Single Molecule Array (Solexa, Inc.) or sequencing-by-synthesis(SBS) utilizing reversible terminator chemistry. These technologies aredescribed in part in U.S. Pat. Nos. 6,969,488; 6,897,023; 6,833,246;6,787,308; and US Publication Application Nos. 20040106110; 20030064398;20030022207; and Constar's, A., The Scientist 2003, 17(13):36.

FIG. 12 illustrates a first embodiment using the SBS approach describedabove.

First, in step 1200 a sample comprising one or more rare cells (e.g.,fetal or CTC) and one or more non-rare cells (e.g., RBC's) is obtainedfrom an animal, such as a human. In step 1202, rare cells, rare DNA(e.g., rare nuclei), or raremRNA is enriched using one or more methodsdisclosed herein or known in the art. Preferably, rare cells areenriched by flowing the sample through an array of obstacles thatselectively directs particles or cells of different hydrodynamic sizesinto different outlets.

In step 1204, enriched genetic material e.g., gDNA is obtained usingmethods known in the art or disclosed herein. In step 1206, the geneticmaterial e.g, gDNA is randomly fragmented. In step 1222, the randomlyfragmented gDNA is ligated with adapters on both ends. In step 1223, thegenetic material, e.g., ssDNA are bound randomly to inside surface of aflow cell channels. In step 1224, unlabeled nucleotides and enzymes areadded to initiate solid phase bridge amplification. The above stepresults in genetic material fragments becoming double stranded and boundat either end to the substrate. In step 1225, the double stranded bridgeis denatured to create to immobilized single stranded genomic DNA (e.g.,ssDNA) sequencing complementary to one another. The above bridgeamplification and denaturation steps are repeated multiple times (e.g.,at least 10, 50, 100, 500, 1,000, 5,000, 10,000, 50,000, 100,000,500,000, 1,000,000, 5,000,000 times) such that several million denseclusters of dsDNA (or immobilized ssDNA pairs complementary to oneanother) are generated in each channel of the flow cell. In step 1226,the first sequencing cycle is initiated by adding all four labeledreversible terminators, primers, and DNA polymerase enzyme to the flowcell. This sequencing-by-synthesis (SBS) method utilizes fourfluorescently labeled modified nucleotides that are especially createdto posses a reversible termination property, which allow each cycle ofthe sequencing reaction to occur simultaneously in the presence of allfour nucleotides (A, C, T, G). In the presence of all four nucleotides,the polymerase is able to select the correct base to incorporate, withthe natural competition between all four alternatives leading to higheraccuracy than methods where only one nucleotide is present in thereaction mix at a time which require the enzyme to reject an incorrectnucleotide. In step 1227, all unincorporated labeled terminators arethen washed off. In step 1228, laser is applied to the flow cell. Laserexcitation captures an image of emitted fluorescence from each clusteron the flow cell. In step 1229, a computer program product comprising acomputer executable logic records the identity of the first base foreach cluster. In step 1230, before initiated the next sequencing step,the 3′ terminus and the fluorescence from each incorporated base areremoved.

Subsequently, a second sequencing cycle is initiated, just as the firstwas by adding all four labeled reversible terminators, primers, and DNApolymerase enzyme to the flow cell. A second sequencing read occurs byapplying a laser to the flow cell to capture emitted fluorescence fromeach cluster on the flow cell which is read and analyzed by a computerprogram product that comprises a computer executable logic to identifythe first base for each cluster. The above sequencing steps are repeatedas necessary to sequence the entire gDNA fragment. In some cases, theabove steps are repeated at least 5, 10, 50, 100, 500, 1,000, 5,000, to10,000 times.

In some embodiments, high-throughput sequencing of mRNA or gDNA can takeplace using AnyDot.chips (Genovoxx, Germany), which allows for themonitoring of biological processes (e.g., mRNA expression or allelevariability (SNP detection). In particular, the AnyDot.chips allow for10×-50× enhancement of nucleotide fluorescence signal detection.AnyDot.chips and methods for using them are described in part inInternational Publication Application Nos. WO 02088382, WO 03020968, WO03031947, WO 2005044836, PCT/EP 05/05657, PCT/EP 05/05655; and GermanPatent Application Nos. DE 101 49 786, DE 102 14 395, DE 103 56 837, DE10 2004 009 704, DE 10 2004 025 696, DE 10 2004 025 746, DE 10 2004 025694, DE 10 2004 025 695, DE 10 2004 025 744, DE 10 2004 025 745, and DE10 2005 012 301.

An overview of one embodiment of the present invention is illustrated inFIG. 13.

First, in step 1300 a sample comprising one or more rare cells (e.g.,fetal, epithelial or CTC) and one or more non-rare cells (e.g., RBC's)is obtained from an animal, such as a human. In step 1302, rare cells orrare genetic material (e.g., gDNA or RNA) is enriched using one or moremethods disclosed herein or known in the art. Preferably, rare cells areenriched by flowing the sample through an array of obstacles thatselectively directs particles or cells of different hydrodynamic sizesinto different outlets. In step 1304, genetic material is obtained fromthe enriched sample. In step 1306, the genetic material (e.g., gDNA) isfragmented into millions of individual nucleic acid molecules and instep 1308, a universal primer binding site is added to each fragment(nucleic acid molecule). In step 1332, the fragments are randomlydistributed, fixed and primed on a surface of a substrate, such as anAnyDot.chip. Distance between neighboring molecules averages 0.1-10 μMor about 1 μm. A sample is applied by simple liquid exchange within amicrofluidic system. Each mm² contains 1 million single DNA moleculesready for sequencing. In step 1334, unbound DNA fragments are removedfrom the substrate; and in step 1336, a solution containing polymeraseand labeled nucleotide analogs having a reversible terminator thatlimits extension to a single base, such as AnyBase.nucleotides areapplied to the substrate. When incorporated into the primer-DNA hybrid,such nucleotide analogs cause a reversible stop of the primer-extension(terminating property of nucleotides). This step represents asingle-base extension. During the stop, incorporated bases, whichinclude a fluorescence label, can be detected on the surface of thesubstrate.

In step 1338, fluorescent dots are detected by a single-moleculefluorescence detection system (e.g., fluorescent microscope). In somecases, a single fluorescence signal (300 nm in diameter) can be properlytracked over the complete sequencing cycles (see below). After detectionof the single-base, in step 1340, the terminating property andfluorescent label of the incorporated nucleotide analogs (e.g.,AnyBase.nucleotides) are removed. The nucleotides are now extendablesimilarly to native nucleotides. Thus, steps 1336-1340 are thusrepeated, e.g., at least 2, 10, 20, 100, 200, 1,000, 2,000 times. Forgenerating sequence data that can be compared with a reference database(for instance human mRNA database of the NCBI), length of the sequencesnippets has to exceed 15-20 nucleotides. Therefore, steps 1 to 3 arerepeated until the majority of all single molecules reaches the requiredlength. This will take, on average, 2 offers of nucleotideincorporations per base.

Other high-throughput sequencing systems include those disclosed inVenter, J., et al. Science 16 Feb. 2001; Adams, M. et al. Science 24Mar. 2000; and M. J. Levene, et al. Science 299:682-686, January 2003;as well as US Publication Application No. 20030044781 and 2006/0078937.Overall such system involve sequencing a target nucleic acid moleculehaving a plurality of bases by the temporal addition of bases via apolymerization reaction that is measured on a molecule of nucleic acid,i.e. the activity of a nucleic acid polymerizing enzyme on the templatenucleic acid molecule to be sequenced is followed in real time. Sequencecan then be deduced by identifying which base is being incorporated intothe growing complementary strand of the target nucleic acid by thecatalytic activity of the nucleic acid polymerizing enzyme at each stepin the sequence of base additions. A polymerase on the target nucleicacid molecule complex is provided in a position suitable to move alongthe target nucleic acid molecule and extend the oligonucleotide primerat an active site. A plurality of labeled types of nucleotide analogsare provided proximate to the active site, with each distinguishabletype of nucleotide analog being complementary to a different nucleotidein the target nucleic acid sequence. The growing nucleic acid strand isextended by using the polymerase to add a nucleotide analog to thenucleic acid strand at the active site, where the nucleotide analogbeing added is complementary to the nucleotide of the target nucleicacid at the active site. The nucleotide analog added to theoligonucleotide primer as a result of the polymerizing step isidentified. The steps of providing labeled nucleotide analogs,polymerizing the growing nucleic acid strand, and identifying the addednucleotide analog are repeated so that the nucleic acid strand isfurther extended and the sequence of the target nucleic acid isdetermined.

In some embodiments, cDNAs, which are reverse transcribed from mRNAsobtained from fetal or maternal cells, are analyzed (e.g. SNP analysisor sequencing) by the methods disclosed herein. The type and abundanceof the cDNAs can be used to determine whether a cell is a fetal cell(such as by the presence of Y chromosome specific transcripts) orwhether the fetal cell has a genetic abnormality (such as aneuploidy,abundance or type of alternative transcripts or problems with DNAmethylation or imprinting).

In one embodiment, fetal or maternal cells are enriched using one ormore methods disclosed herein. Preferably, fetal cells are enriched byflowing the sample through an array of obstacles that selectivelydirects particles or cells of different hydrodynamic sizes intodifferent outlets such that fetal cells and cells larger than fetalcells are directed into a first outlet and one or more cells orparticles smaller than the rare cells are directed into a second outlet.

Total. RNA or poly-A mRNA is then obtained from enriched cell(s) (fetalor maternal cells) using purification techniques known in the art.Generally, about 1 μg-2 μg of total RNA is sufficient. Next, afirst-strand complementary DNA (cDNA) is synthesized using reversetranscriptase and a single T7-oligo(dT) primer. Next, a second-strandcDNA is synthesized using DNA ligase, DNA polymerase, and RNase enzyme.Next, the double stranded cDNA (ds-cDNA) is purified.

Analyzing the rare cells to determine the existence of condition ordisease may also include detecting mitochondrial DNA, telomerase, or anuclear matrix protein in the enriched rare cell sample; detecting thepresence or absence of perinuclear compartments in a cell of theenriched sample; or performing gene expression analysis, determiningnucleic acid copy number, in-cell PCR, or fluorescence in-situhybridization of the enriched sample.

In some embodiments, PCR-amplified single-strand nucleic acid ishybridized to a primer and incubated with a polymerase, ATP sulfurylase,luciferase, apyrase, and the substrates luciferin and adenosine 5′phosphosulfate. Next, deoxynucleotide triphosphates corresponding to thebases A, C, G, and T (U) are added sequentially. Each base incorporationis accompanied by release of pyrophosphate, converted to ATP bysulfurylase, which drives synthesis of oxyluciferin and the release ofvisible light. Since pyrophosphate release is equimolar with the numberof incorporated bases, the light given off is proportional to the numberof nucleotides adding in any one step. The process repeats until theentire sequence is determined. In one embodiment, pyrosequencinganalyzes DNA methylations, mutation and SNPs. In another embodiment,pyrosequencing also maps surrounding sequences as an internal qualitycontrol. Pyrosequencing analysis methods are known in the art.

In some embodiments, sequence analysis of the rare cell's geneticmaterial may include a four-color sequencing by ligation scheme(degenerate ligation), which involves hybridizing an anchor primer toone of four positions. Then an enzymatic ligation reaction of the anchorprimer to a population of degenerate nonamers that are labeled withfluorescent dyes is performed. At any given cycle, the population ofnonamers that is used is structure such that the identity of one of itspositions is correlated with the identity of the fluorophore attached tothat nonamer. To the extent that the ligase discriminates forcomplementarity at that queried position, the fluorescent signal allowsthe inference of the identity of the base. After performing the ligationand four-color imaging, the anchor primer:nonamer complexes are strippedand a new cycle begins. Methods to image sequence information afterperforming ligation are known in the art.

Another embodiment includes kits for performing some or all of the stepsof the invention. The kits may include devices and reagents in anycombination to perform any or all of the steps. For example, the kitsmay include the arrays for the size-based separation or enrichment, thedevice and reagents for magnetic separation and the reagents needed forthe genetic analysis.

EXAMPLES Example 1 Separation of Fetal Cord Blood

FIGS. 14A-14D shows a schematic of the device used to separate nucleatedcells from fetal cord blood.

Dimensions: 100 mm×28 mm×1 mm

Array design: 3 stages, gap size=18, 12 and 8 μm for the first, secondand third stage, respectively.

Device fabrication: The arrays and channels were fabricated in siliconusing standard photolithography and deep silicon reactive etchingtechniques. The etch depth is 140 μm. Through holes for fluid access aremade using KOH wet etching. The silicon substrate was sealed on theetched face to form enclosed fluidic channels using a blood compatiblepressure sensitive adhesive (9795, 3M, St Paul, Minn.).

Device packaging: The device was mechanically mated to a plasticmanifold with external fluidic reservoirs to deliver blood and buffer tothe device and extract the generated fractions.

Device operation: An external pressure source was used to apply apressure of 2.0 PSI to the buffer and blood reservoirs to modulatefluidic delivery and extraction from the packaged device.

Experimental conditions: Human fetal cord blood was drawn into phosphatebuffered saline containing Acid Citrate Dextrose anticoagulants. 1 mL ofblood was processed at 3 mL/hr using the device described above at roomtemperature and within 48 hrs of draw. Nucleated cells from the bloodwere separated from enucleated cells (red blood cells and platelets),and plasma delivered into a buffer stream of calcium and magnesium-freeDulbecco's Phosphate Buffered Saline (14190-144, Invitrogen, Carlsbad,Calif.) containing 1% Bovine Serum Albumin (BSA) (A8412-100ML,Sigma-Aldrich, St Louis, Mo.) and 2 mM EDTA (15575-020, Invitrogen,Carlsbad, Calif.).

Measurement techniques: Cell smears of the product and waste fractions(FIG. 15A-15B) were prepared and stained with modified Wright-Giemsa(WG16, Sigma Aldrich, St. Louis, Mo.).

Performance: Fetal nucleated red blood cells were observed in theproduct fraction (FIG. 15A) and absent from the waste fraction (FIG.15B).

Example 2 Isolation of Fetal Cells from Maternal blood

The device and process described in detail in Example 1 were used incombination with immunomagnetic affinity enrichment techniques todemonstrate the feasibility of isolating fetal cells from maternalblood.

Experimental conditions: blood from consenting maternal donors carryingmale fetuses was collected into K₂EDTA vacutainers (366643, BectonDickinson, Franklin Lakes, N.J.) immediately following electivetermination of pregnancy. The undiluted blood was processed using thedevice described in Example 1 at room temperature and within 9 hrs ofdraw. Nucleated cells from the blood were separated from enucleatedcells (red blood cells and platelets), and plasma delivered into abuffer stream of calcium and magnesium-free Dulbecco's PhosphateBuffered Saline (14190-144, Invitrogen, Carlsbad, Calif.) containing 1%Bovine Serum Albumin (BSA) (A8412-100ML, Sigma-Aldrich, St Louis, Mo.).Subsequently, the nucleated cell fraction was labeled with anti-CD71microbeads (130-046-201, Miltenyi Biotech Inc., Auburn, Calif.) andenriched using the MiniMACS™ MS column (130-042-201, Miltenyi BiotechInc., Auburn, Calif.) according to the manufacturer's specifications.Finally, the CD71-positive fraction was spotted onto glass slides.

Measurement techniques: Spotted slides were stained using fluorescencein situ hybridization (FISH) techniques according to the manufacturer'sspecifications using Vysis probes (Abbott Laboratories, Downer's Grove,Ill.). Samples were stained from the presence of X and Y chromosomes. Inone case, a sample prepared from a known Trisomy 21 pregnancy was alsostained for chromosome 21.

Performance: Isolation of fetal cells was confirmed by the reliablepresence of male cells in the CD71-positive population prepared from thenucleated cell fractions (FIG. 16). In the single abnormal case tested,the trisomy 21 pathology was also identified (FIG. 17).

Example 3 Confirmation of the Presence of Male Fetal Cells in EnrichedSamples

Confirmation of the presence of a male fetal cell in an enriched sampleis performed using qPCR with primers specific for DYZ, a marker repeatedin high copy number on the Y chromosome. After enrichment of fnRBC byany of the methods described herein, the resulting enriched fnRBC arebinned by dividing the sample into 100 PCR wells. Prior to binning,enriched samples may be screened by FISH to determine the presence ofany fnRBC containing an aneuploidy of interest. Because of the lownumber of fnRBC in maternal blood, only a portion of the wells willcontain a single fnRBC (the other wells are expected to be negative forfnRBC). The cells are fixed in 2% Paraformaldehyde and stored at 4° C.Cells in each bin are pelleted and resuspended in 5 μl PBS plus 1 μl 20mg/ml Proteinase K (Sigma #P-2308). Cells are lysed by incubation at 65°C. for 60 minutes followed by inactivation of the Proteinase K byincubation for 15 minutes at 95° C. For each reaction, primer sets (DYZforward primer TCGAGTGCATTCCATTCCG; DYZ reverse primerATGGAATGGCATCAAACGGAA; and DYZ Taqman Probe6FAM-TGGCTGTCCATTCCA-MGBNFQ), TaqMan Universal PCR master mix, NoAmpErase and water are added. The samples are run and analysis isperformed on an ABI 7300: 2 minutes at 50° C., 10 minutes 95° C.followed by 40 cycles of 95° C. (15 seconds) and 60° C. (1 minute).Following confirmation of the presence of male fetal cells, furtheranalysis of bins containing fnRBC is performed. Positive bins may bepooled prior to further analysis.

FIG. 27 shows the results expected from such an experiment. The data inFIG. 27 was collected by the following protocol. Nucleated red bloodcells were enriched from cord cell blood of a male fetus by sucrosegradient two Heme Extractions (HE). The cells were fixed in 2%paraformaldehyde and stored at 4° C. Approximately 10×1000 cells werepelleted and resuspended each in 5 μl PBS plus 1 μl 20 mg/ml ProteinaseK (Sigma #P-2308). Cells were lysed by incubation at 65° C. for 60minutes followed by a inactivation of the Proteinase K by 15 minute at95° C. Cells were combined and serially diluted 10-fold in PBS for 100,10 and 1 cell per 6 μl final concentration were obtained. Six μl of eachdilution was assayed in quadruplicate in 96 well format. For eachreaction, primer sets (DYZ forward primer TCGAGTGCATTCCATTCCG; 0.9 uMDYZ reverse primer ATGGAATGGCATCAAACGGAA; and 0.5 uM DYZ TaqMan Probe6FAM-TGGCTGTCCATTCCA-MGBNFQ), TaqMan Universal PCR master mix, NoAmpErase and water were added to a final volume of 25 μl per reaction.Plates were run and analyzed on an ABI 7300: 2 minutes at 50° C., 10minutes 95° C. followed by 40 cycles of 95° C. (15 seconds) and 60° C.(1 minute). These results show that detection of a single fnRBC in a binis possible using this method.

Example 4 Confirmation of the Presence of Fetal Cells in EnrichedSamples by STR Analysis

Maternal blood is processed through a size-based separation module, withor without subsequent MHEM enhancement of fnRBCs. The enhanced sample isthen subjected to FISH analysis using probes specific to the aneuploidyof interest (e.g., triploidy 13, triploidy 18, and XYY). Individualpositive cells are isolated by “plucking” individual positive cells fromthe enhanced sample using standard micromanipulation techniques. Using anested PCR protocol, STR marker sets are amplified and analyzed toconfirm that the FISH-positive aneuploid cell(s) are of fetal origin.For this analysis, comparison to the maternal genotype is typical. Anexample of a potential resulting data set is shown in Table 2.Non-maternal alleles may be proven to be paternal alleles by paternalgenotyping or genotyping of known fetal tissue samples. As can be seen,the presence of paternal alleles in the resulting cells, demonstratesthat the cell is of fetal origin (cells #1, 2, 9, and 10). Positivecells may be pooled for further analysis to diagnose aneuploidy of thefetus, or may be further analyzed individually.

TABLE 2 STR locus alleles in maternal and fetal cells STR STR locus STRlocus STR locus locus STR locus DNA Source D14S D16S D8S F13B vWAMaternal alleles 14, 17 11, 12 12, 14 9, 9 16, 17 Cell #1 alleles  8 19Cell #2 alleles 17 15 Cell #3 alleles 14 Cell #4 alleles Cell #5 alleles17 12 9 Cell #6 alleles Cell #7 alleles 19 Cell #8 alleles Cell #9alleles 17 14 7, 9 17, 19 Cell #10 alleles 15

Example 5 Confirmation of the Presence of Fetal Cells in EnrichedSamples by SNP Analysis

Maternal blood is processed through a size-based separation module, withor without subsequent MHEM enhancement of fnRBCs. The enhanced sample isthen subjected to FISH analysis using probes specific to the aneuploidyof interest (e.g., triploidy 13, triploidy 18, and XYY). Samples testingpositive with FISH analysis are then binned into 96 microtiter wells,each well containing 15 μl of the enhanced sample. Of the 96 wells, 5-10are expected to contain a single fnRBC and each well should containapproximately 1000 nucleated maternal cells (both WBC and mnRBC). Cellsare pelleted and resuspended in 5 μl PBS plus 1 μl 20 mg/ml Proteinase K(Sigma #P-2308), Cells are lysed by incubation at 65° C. for 60 minutesfollowed by a inactivation of the Proteinase K by 15 minute at 95° C.

In this example, the maternal genotype (BB) and fetal genotype (AB) fora particular set of SNPs is known. The genotypes A and B encompass allthree SNPs and differ from each other at all three SNPs. The followingsequence from chromosome 7 contains these three SNPs (rs7795605,rs7795611 and rs7795233 indicated in brackets, respectively)ATGCAGCAAGGCACAGACTAA[G/A]CAAGGAGA[G/C]GCAAAATTTTC[A/G]TAGGGGAGAGAAATGGGTCATT).

In the first round of PCR, genomic DNA from binned enriched cells isamplified using primers specific to the outer portion of thefetal-specific allele A and which flank the interior SNP (forward primerATGCAGCAAGGCACAGACTACG; reverse primer AGAGGGGAGAGAAATGGGTCATT). In thesecond round of PCR, amplification using real time SYBR Green PCR isperformed with primers specific to the inner portion of allele A andwhich encompass the interior SNP (forward primerCAAGGCACAGACTAAGCAAGGAGAG; reverse primerGGCAAAATTTTCATAGGGGAGAGAAATGGGTCATT).

Expected results are shown in FIG. 28. Here, six of the 96 wells testpositive for allele A, confirming the presence of cells of fetal origin,because the maternal genotype (BB) is known and cannot be positive forallele A. DNA from positive wells may be pooled for further analysis oranalyzed individually.

Example 6 Amplification and Sequencing of STRs for Fetal Diagnosis

Fetal cells or nuclei can be isolated as describe in the enrichmentsection or as described in example 1 and 2. DNA from the fetal cells orisolated nuclei from fetal cells can be obtained using any methods knownin the art. STR loci can be chosen on the suspected trisomic chromosomes(X, 13, 18, or 21) and on other control chromosomes. These would beselected for high heterozygosity (variety of alleles) so that thepaternal allele of the fetal cells is more likely to be distinct inlength from the maternal alleles, with resulting improved power todetect. Di-, tri-, or tetra-nucleotide repeat loci can be used. The STRloci can then be amplified according the methods described in theamplification section.

For instance, the genomic DNA from the enriched fetal cells and amaternal control sample can be fragmented, and separated into singlestrands. The single strands of the target nucleic acids would be boundto beads under conditions that favor each single strand molecule of DNAto bind a different bead. Each bead would then be captured within adroplet of a PCR-reaction-mixture-in-oil-emulsion and PCR amplificationoccurs within each droplet. The amplification on the bead could resultsin each bead carrying at least one 10 million copies of the uniquesingle stranded target nucleic acid. The emulsion would be broken, theDNA is denatured and the beads carrying single-stranded nucleic acidsclones would be deposited into a picolitre-sized well for furtheranalysis.

The beads can then be placed into a highly parallel sequencing bysynthesis machine which can generate over 400,000 reads (˜100 bp perread) in a single 4 hour run. Sequence by synthesis involves inferringthe sequence of the template by synthesizing a strand complementary tothe target nucleic acid sequence. The identity of each nucleotide wouldbe detected after the incorporation of a labeled nucleotide ornucleotide analog into a growing strand of a complementary nucleic acidsequence in a polymerase reaction. After the successful incorporation ofa label nucleotide, a signal would be measured and then nulled and theincorporation process would be repeated until the sequence of the targetnucleic acid is identified. The allele abundances for each of the STRsloci can then be determined. The presence of trisomy would be determinedby comparing abundance for each of the STR loci in the fetal cells withthe abundance for each of the SRTs loci in a maternal control sample.The enrichment, amplification and sequencing methods described in thisexample allow for the analysis of rare alleles from fetal cells, even incircumstances where fetal cells are in a mixed sample comprising othermaternal cells, and even in circumstances where other maternal cellsdominate the mixture.

Example 7 Analysis of STR's Using Quantitative Fluorescence

Genomic DNA from enriched fetal cells and a maternal control sample willbe genotyped for specific SIR loci in order to assess the presence ofchromosomal abnormalities, such as trisomy. Due to the small number offetal cells typically isolated from maternal blood it is advantageous toperform a pre-amplification step prior to analysis, using a protocolsuch as improved primer extension pre-amplification (IPEP) PCR. Celllysis is carried out in 10 ul High Fidelity buffer (50 mM Tris-HCL, 22mM (NH.sub.4).sub.2 SO.sub.4 2.5 mM MgCl.sub.2, pH 8.9) which alsocontained 4 mg/ml proteinase K and 0.5 vol % Tween 20 (Merck) for 12hours at 48° C. The enzyme is then inactivated for 15 minutes at 94° C.Lysis is performed in parallel batches in 5 ul, 200 mM KOH, 50 mMdithiothreitol for 10 minutes at 65.degree. The batches are thenneutralized with 5 ul 900 mM TrisHCl pH 8.3, 300 mM KCl. Preamplicationis then carried out for each sample using completely randomized 15-merprimers (16 uM) and dNTP (100 uM) with 5 units of a mixture of Taqpolymerase (Boehringer Mannheim) and Pwo polymerase (BoehringerMannheim) in a ratio of 10:1 under standard PCR buffer conditions (50 mMTris-HCL, 22 mM (NH₄)₂ SO₄, 2.5 mM Mg₂, pH 8.9, also containing 5% byvol. of DMSO) in a total volume of 60 ul with the following 50 thermalcycles: Step Temperature Time (1) 92° C. 1 Min 30 Sec; (2) 92° C. 40 Min(3) 37° C. 2 Min; (4) ramp: 0.1° C./sec to 55° C. (5) 55° C. 4 Min (6)68° C. 30 Sec (7) go to step 2, 49 times (8) 8° C. 15′Min.

Dye labeled primers are then chosen from Table 3 based on STR loci onchromosomes of interest, such as 13, 18, 21 or X. The primers aredesigned so that one primer of each pair contains a fluorescent dye,such as ROX, HEX, JOE, NED, FAM, TAMARA or LIZ. The primers are placedinto multiplex mixes based on expected product size, fluorescent tagcompatibility and melting temperature. This allows multiple SIR loci tobe assayed at once and yet still conserves the amount of initialstarting material required. All primers are initially diluted to aworking dilution of 10 pM. The primers are then combined in a cocktailthat has a final volume of 40 ul. Final primer concentration isdetermined by reaction optimization. Additional PCR grade water is addedif the primer mix is below 40 ul. A reaction mix containing 6 ul ofSigma PCR grade water, 1.25 ul of Perkin Elmer Goldamp PCR buffer, 0.5ul of dNTPs, 8 ul of the primer cocktail, 0.12 ul of Perkin Elmer TaqGold Polymerase and 1.25 ul of Mg (25 mM) is mixed for each sample. Tothis a 1 ul sample containing preamplified DNA from enriched fetal cellsor maternal control genomic DNA is added.

The reaction mix is amplified in a DNA thermocyler, (PTC-200; MJResearch) using an amplification cycle optimized for the meltingtemperature of the primers and the amount of sample DNA.

The amplification product will then analyzed using an automated DNAsequencer system, such as the ABI 310, 377, 3100, 3130, 3700 or 3730, orthe Li-Cor 4000, 4100, 4200 or 4300. For example when the amplificationproducts are prepared for analysis on a ABI 377 sequencer, 0.6 ul ofproducts will be removed and combined with 1.6 ul of loading buffer mix.The master loading buffer mix contains 90 ul deionized formamidecombined with 25 ul Perkin Elmer loading dye and 10 ul of a sizestandard, such as the ROX 350 size standard. Various other standards canbe used interchangeably depending on the sizes of the labeled PCRproducts. The loading buffer and sample are then heat denatured at 95°C. for 3 minutes followed by flash cooling on ice. 2 ul of theproduct/buffer mix is then electrophoresed on a 12 inch 6% (19:1)polyacrylamide gel on an ABI 377 sequencer.

The results are then analyzed using ABI Genotyper software. Theincorporation of a fluorochrome during amplification allows productquantification for each chromosome specific STR, with 2 fluorescentpeaks observed in a normal heterozygous individual with an approximateratio of 1:1. By comparison in trisomic samples, either 3 fluorescentpeaks with a ratio of 1:1:1 (trialleleic) or 2 peaks with a ratio ofaround 2:1 (diallelic) are observed. Using this method screening may becarried out for common trisomies and sex chromosome aneuploidy in asingle reaction.

TABLE 3 Primer Sets for STRs on Chromsomes 13, 18, 21 and X Ch.STR Marker Primer 1 Primer 2 13 D1353175ACAGAAGTCTGOGATGTGGA (SEQ ID NO 1) GCCCAAAAAGACAGACAGAA (SEQ ID NO 2)D13S1493 ACCTGTTGTATGGCAGCAGT (SEQ ID NO 3)AGTTGACTCTTTCCCCAACTA (SEQ ID NO 4) D1351807TTTGGTAAGAAAAACATCTCCC (SEQ ID NO 5) GGCTGCAGTTAGCTGTCATT (SEQ ID NO 6)D13S256 CCTGGGCAACAAGAGCAAA (SEQ ID NO 7)AGCAGAGAGACATAATTGTG (SEQ ID NO 8) D13S258-ACCTGCCAAATTTTACCAGG (SEQ ID NO 9) GACAGAGAGAGGGAATAAACC (SEQ ID NO 10)D13S285 ATATATGCACATCCATCCATG (SEQ ID NO 11) GGCCAAAGATAGATAGCAAGGTA(SEQ ID NO 12) D13S303 ACATCGCTCCTTACCCCATC (SEQ ID NO 13)TGTACCCATTAACCATCCCCA (SEQ ID NO 14) D13S317ACAGAAGTCTGGGATGTGGA (SEQ ID NO 15) GCCCAAAAAGACAGACAGAA (SEQ ID NO 16)D135779 AGAGTGAGATTCTGTCTCAATTAA GGCCCTGTGTAGAAGCTGTA (SEQ ID NO 18)(SEQ ID NO 17) D13S787 ATCAGGATTCCAGGAGGAAA (SEQ ID NO 19)ACCTGGGAGGCGGAGCTC (SEQ ID NO 20) D13S793 GGCATAAAAATAGTACAGCAAGCATTTGAACAGAGGCATGTAC (SEQ ID NO 22) (SEQ ID NO 21) D13S796CATGGATGCAGAATTCACAG (SEQ ID NO 23) TCATCTCCCTGTTTGGTAGC (SEQ ID NO 24)DI3S800 AGGGATCTTCAGAGAAACAGG (SEQ ID NO 25)TGACACTATCAGCTCTCTGGC (SEQ ID NO 26) D13S894 GGTGCTTGCTGTAAATATAATTGCACTACAGCAGATTGCACCA (SEQ ID NO 28) (SEQ ID NO 27) 18 D18S51CAAACCCGACTACCAGCAAC (SEQ ID NO 29) GAGCCATGTTCATGCCACTG (SEQ ID NO 30)D18S1002 CAAAGAGTGAATGCTGTACAAACAGC CAAGATGTGAGTGTGCTTTTCAGGAG(SEQ ID NO 31) (SEQ ID NO 32) D1851357ATCCCACAGGATGCCTATTT (SEQ ID NO 33) ACGGGAGCTTTTGAGAAGTT (SEQ ID NO 34)D18S1364 TCAAATTTTTAAGTCTCACCAGG GCCTGTAGAAAGCAACAACC (SEQ ID NO 36)(SEQ ID NO 35) D18S1370 GGTGACAGAGCAAGACCTTG (SEQ ID NO 37)GCCTCTTGTCATCCCAAGTA (SEQ ID NO 38) D18S1371CTCTCTTCATCCACCATTGG (SEQ ID NO 39) GCTGTAAGAGACCTGTGTTG (SEQ ID NO 40)D18S1376 TGGAACCACTTCATTCTTGG (SEQ ID NO 41)ATTTCAGACCAAGATAGGC (SEQ ID NO 42) D18S1390CCTATTTAAGTTTCTGTAAGG (SEQ ID NO 43) ATGGTGTAGACCCTGTGGAA (SEQ ID NO 44)D18S499 CTGCACAACATAGTGAGACCTG (SEQ ID NO 45) AGATTACCCAGAAATGAGATCAGC(SEQ ID NO 46) D18S535 TCATGTGACAAAAGCCACAC (SEQ ID NO 47)AGACAGAAATATAGATGAGAATGCA (SEQ ID NO 48) D18S535TCATGTGACAAAAGCCACAC (SEQ ID NO 49) AGACAGAAATATAGATGAGAATGCA(SEQ ID NO 50) D18S542 TTTCCAGTGGAAACCAAACT (SEQ ID NO 51)TCCAGCAACAACAAGAGACA (SEQ ID NO 52) D18S843GTCCTCATCCTGTAAAACGGG (SEQ ID NO 53) CCACTAACTAGTTTGTGACTTTGG(SEQ ID NO 54) D18S851 CTGTCCTCTAGGCTCATTTAGC (SEQ ID NO 55)TTATGAAGCAGTGATGCCAA (SEQ ID NO 56) D18S858AGCTGGAGAGGGATAGCATT (SEQ ID NO 57) TGCATTGCATGAAAGTAGGA (SEQ ID NO 58)D18S877 GATGATAGAGATGGCACATGA (SEQ ID NO 59) TCTTCATACATGCTTTATCATGC(SEQ ID NO 60) 21 D21S11 GTGAGTCAATTCCCCAAG (SEQ ID NO 61)GTTGTATTAGTCAATGTTCTCC (SEQ ID NO 62) D2151411ATGATGAATGCATAGATGGATG (SEQ ID NO 63) AATGTGTGTCCTTCCAGGC (SEQ ID NO 64)D21S1413 TTGCAGGGAAACCACAGTT (SEQ ID NO 65)TCCTTGGAATAAATTCCCGG (SEQ ID NO 66) D21S1432 CTTAGAGGGACAGAACTAATAGGCAGCCTATTGTGGGTTTGTGA (SEQ ID NO 68) (SEQ ID NO 67) D21S1437ATGTACATGTGTCTGGGAAGG (SEQ ID NO 69) TTCTCTACATATTTACTGCCAACA(SEQ ID NO 70) D21S1440 GAGTTTGAAAATAAAGTGTTCTGCCCCCACCCCTTTTAGTTTTA (SEQ ID NO 72) (SEQ ID NO 71) D21S1446ATGTACGATACGTAATACTTGACAA GTCCCAAAGGACCTGCTC (SEQ ID NO 74)(SEQ ID NO 73) D21S2052 GCACCCCTTTATACTTGGGTG (SEQ ID NO 75)TAGTACTCTACCATCCATCTATCCC (SEQ ID NO 76) D21S2055AACAGAACCAATAGGCTATCTATC TACAGTAAATCACTTGGTAGGAGA (SEQ ID NO 77)(SEQ ID NO 78) X SBMA TCCGCGAAGTGAAGAAC (SEQ ID NO 79)CTTGGGGAGAACCATCCTCA (SEQ ID NO 80) DXS1047CCGGCTACAAGTGATGTCTA (SEQ ID NO 81) CCTAGGTAACATAGTGAGACCTTG(SEQ ID NO 82) DXS1068 CCTCTAAAGCATAGGGTCCA (SEQ ID NO 83)CCCATCTGAGAACACGCTG (SEQ ID NO 84) DXS1283E AGTTTAGGAGATTATCAAGCTGGGTTCCCATAATAGATGTATCCAG (SEQ ID NO 85) (SEQ ID NO 86) DXS6789TTGGTACTTAATAAACCCTCTTTT CTAGAGGGACAGAACCAATAGG (SEQ 1D NO 87)(SEQ ID NO 88) DXS6795 TGTCTGCTAATGAATGATTTGG (SEQ ID NO 89)CCATCCCCTAAACCTCTCAT (SEQ ID NO 90) DXS6800GTGGGACCTTGTGATTGTGT (SEQ ID NO 91) CTGGCTGACACTTAGGGAAA (SEQ ID NO 92)DXS6810 ACAGAAAACCTTTTGGGACC (SEQ ID NO 93)CCCAGCCCTGAATATTATCA (SEQ ID NO 94) DXS7127TGCACTTAATATCTGGTGATGG (SEQ ID NO 95)ATTTCTTTCCCTCTGCAACC (SEQ ID NO 96) DXS7132AGCCCATTTTCATAATAAATCC (SEQ ID NO 97) AATCAGTGCTTTCTGTACTATTGG(SEQ 1D NO 98) DXS8377 CACTTCATGGCTTACCACAG (SEQ ID NO 99)GACCTTTGGAAAGCTAGTGT (SEQ ID NO 100) DXS9893TGTCACGTTTACCCTGGAAC (SEQ ID NO 101) TA TTCTTCTATCCAACCAACAGC(SEQ ID NO 102) DXS9895 TTGGGTGGGGACACAGAG (SEQ ID NO 103)CCTGGCTCAAGGAATTACAA (SEQ ID NO 104) DXS9896CCAGCCTGGCTGTTAGAGTA (SEQ D NO 105) ATATTCTTATATTCCATATGGCACA(SEQ ID NO 106) DXS9902 TGGAGTCTCTGGGTGAAGAG (SEQ ID NO 107)CAGGAGTATGGGATCACCAG (SEQ ID NO 108) DXS998CAGCAATTTTTCAAAGGC (SEQ ID NO 109) AGATCATTCATATAACCTCAAAAGA(SEQ ID NO 110)

Example 8 Detection of Mutations Related to Fetal Abnormalities

Fetal cells or nuclei can be isolated as describe in the Enrichmentsection or as described in example 1 and 2. DNA from the fetal cells orisolated nuclei from fetal cells can be obtained using any methods knownin the art. The presence of mutations of DNA or RNA from the geneslisted in FIG. 4 can then be analyzed. DNA or RNA of any of the geneslisted in table 3 can then be amplified according the methods describedin the amplification section.

For instance, the genomic DNA from the enriched fetal cells and amaternal control sample can be fragmented, and separated into singlestrands. The single strands of the target nucleic acids would be boundto beads under conditions that favor each single strand molecule of DNAto bind a different bead. Each bead would then be captured within adroplet of a PCR-reaction-mixture-in-oil-emulsion and PCR amplificationoccurs within each droplet. The amplification on the bead could resultsin each bead carrying at least one 10 million copies of the uniquesingle stranded target nucleic acid. The emulsion would be broken, theDNA would be denatured and the beads carrying single-stranded nucleicacids clones would be deposited into a picolitre-sized well for furtheranalysis.

The beads can then be placed into a highly parallel sequencing bysynthesis machine which can generate over 400,000 reads (˜100 bp perread) in a single 4 hour run. Sequence by synthesis involves inferringthe sequence of the template by synthesizing a strand complementary tothe target nucleic acid sequence. The identity of each nucleotide wouldbe detected after the incorporation of a labeled nucleotide ornucleotide analog into a growing strand of a complementary nucleic acidsequence in a polymerase reaction. After the successful incorporation ofa label nucleotide, a signal would be measured and then nulled and theincorporation process would be repeated until the sequence of the targetnucleic acid is identified. The presence of a mutation can then bedetermined. The enrichment, amplification and sequencing methodsdescribed in this example allow for the analysis of rare nucleic acidsfrom fetal cells, even in circumstances where fetal cells are in a mixedsample comprising other maternal cells and even in circumstances wherematernal cells dominate the mixture.

Example 9 Quantitative Genotyping Using Molecular Inversion Probes forTrisomy Diagnosis on Fetal Cells

Fetal cells or nuclei can be isolated as described in the enrichmentsection or as described in example 1 and 2. Quantitative genotyping canthen be used to detect chromosome copy number changes. The output of theenrichment procedure would be divided into separate wells of amicrotiter plate with the number of wells chosen so no more than onecell or genome copy is located per well, and where some wells may haveno cell or genome copy at all.

Perform multiplex PCR and Genotyping using MIP technology with binspecific tags: PCR primer pairs for multiple (40-100) highly polymorphicSNPs can then be added to each well in the microtiter plate. Forexample, SNPs primers can be designed along chromosomes 13, 18, 21 and Xto detect the most frequent aneuploidies, and along control regions ofthe genome where aneuploidy is not expected. Multiple (˜10) SNPs wouldbe designed for each chromosome of interest to allow for non-informativegenotypes and to ensure accurate results. PCR primers would be chosen tobe multiplexible with other pairs (fairly uniform melting temperature,absence of cross-priming on the human genome, and absence ofprimer-primer interaction based on sequence analysis). The primers wouldbe designed to generate amplicons 70-100 bp in size to increase theperformance of the multiplex PCR. The primers would contain a 22 bp tagon the 5′ which is used in the genotyping analysis. A second of round ofPCR using nested primers may be performed to ensure optimal performanceof the multiplex amplification.

The Molecular Inversion Probe (MIP) technology developed by Affymetrix(Santa Clara, Calif.) can genotype 20,000 SNPs or more in a singlereaction. In the typical MIP assay, each SNP would be assigned a 22 bpDNA tag which allows the SNP to be uniquely identified during the highlyparallel genotyping assay. In this example, the DNA tags serve tworoles: 1) determine the identity of the different SNPs and 2) determinethe identity of the well from which the genotype was derived.

The tagged MIP probes would be combined with the amplicons from theinitial multiplex single-cell PCR and the genotyping reactions would beperformed. The probe/template mix would be divided into 4 tubes eachcontaining a different nucleotide (e.g. G, A, T or C). Following anextension and ligation step, the mixture would be treated withexonuclease to remove all linear molecules and the tags of the survivingcircular molecules would be amplified using PCR. The amplified tags formall of the bins would then be pooled and hybridized to a single DNAmicroarray containing the complementary sequences to each of the 20,000tags.

Identify bins with non-maternal alleles (e.g. fetal cells): The firststep in the data analysis procedure would be to use the 22 bp tags tosort the 20,000 genotypes into bins which correspond to the individualwells of the original microtiter plates. The second step would be toidentify bins contain non-maternal alleles which correspond to wellsthat contained fetal cells. Determining the number bins withnon-maternal alleles relative to the total number of bins would providean accurate estimate of the number of fnRBCs that were present in theoriginal enriched cell population. When a fetal cell is identified in agiven bin, the non-maternal alleles would be detected by 40 independentSNPs which provide an extremely high level of confidence in the result.

Detect aneuploidy for chromosomes 13, 18, and 21: After identifyingapproximately 10 bins that contain fetal cells, the next step would beto determine the ploidy of chromosomes 13, 18, 21 and X by comparingratio of maternal to paternal alleles for each of the 10 SNPs on eachchromosome. The ratios for the multiple SNPs on each chromosome can becombined (averaged) to increase the confidence of the aneuploidy callfor that chromosome. In addition, the information from the approximate10 independent bins containing fetal cells can also be combined tofurther increase the confidence of the call.

Example 10 Fetal Diagnosis with CGH

Fetal cells or nuclei can be isolated as described in the enrichmentsection or as described in example 1 and 2. Comparative genomichybridization (CGH) can be used to determine copy numbers of genes andchromosomes. DNA extracted from the enriched fetal cells will behybridized to immobilized reference DNA which can be in the form ofbacterial artificial chromosome (BAC) clones, or PCR products, orsynthesized DNA oligos representing specific genomic sequence tags.Comparing the strength of hybridization fetal cells and maternal controlcells to the immobilized DNA segments gives a copy number ratio betweenthe two samples. To perform CGH effectively starting with small numbersof cells, the DNA from the enriched fetal cells can be amplifiedaccording to the methods described in the amplification section.

A ratio-preserving amplification of the DNA would be done to minimizethese errors; i.e. this amplification method would be chosen to produceas close as possible the same amplification factor for all targetregions of the genome. Appropriate methods would include multipledisplacement amplification, the two-stage PCR, and linear amplificationmethods such as in vitro transcription.

To the extent the amplification errors are random their effect can bereduced by averaging the copy number or copy number ratios determined atdifferent loci over a genomic region in which aneuploidy is suspected.For example, a microarray with 1000 oligo probes per chromosome couldprovide a chromosome copy number with error bars ˜sqrt(1000) timessmaller than those from the determination based on a single probe. It isalso important to perform the probe averaging over the specific genomicregion(s) suspected for aneuploidy. For example, a common knownsegmental aneuploidy would be tested for by averaging the probe dataonly over that known chromosome region rather than the entirechromosome. Segmental aneuploidies can be caused by a chromosomalrearrangement, such as a deletion, duplication or translocation event.Random errors could be reduced by a very large factor using DNAmicroarrays such as Affymetrix arrays that could have a million or moreprobes per chromosome.

In practice other biases will dominate when the random amplificationerrors have been averaged down to a certain level, and these biases inthe CGH experimental technique must be carefully controlled. Forexample, when the two biological samples being compared are hybridizedto the same array, it is helpful to repeat the experiment with the twodifferent labels reversed and to average the two results—this techniqueof reducing the dye bias is called a ‘fluor reversed pair’. To someextent the use of long ‘clone’ segments, such as BAC clones, as theimmobilized probes provides an analog averaging of these kinds oferrors; however, a larger number of shorter oligo probes should besuperior because errors associated with the creation of the probefeatures are better averaged out.

Differences in amplification and hybridization efficiency from sequenceregion to sequence region may be systematically related to DNA sequence.These differences can be minimized by constraining the choices of probesso that they have similar melting temperatures and avoid sequences thattend to produce secondary structure. Also, although these effects arenot truly ‘random’, they will be averaged out by averaging the resultsfrom a large number of array probes. However, these effects may resultin a systematic tendency for certain regions or chromosomes to haveslightly larger signals than others, after probe averaging, which maymimic aneuploidy. When these particular biases are in common between thetwo samples being compared, they divide out if the results arenormalized so that control genomic regions believed to have the samecopy number in both samples yield a unity ratio.

After performing CGH analysis trisomy can be diagnosed by comparing thestrength of hybridization fetal cells and maternal control cells to theimmobilized DNA segments which would give a copy number ratio betweenthe two samples.

In one method, DNA samples will be obtained from the genomic DNA fromenriched fetal cells and a maternal control sample. These samples aredigested with the Alu I restriction enzyme (Promega, catalog #R6281) inorder to introduce nicks into the genomic DNA (e.g. 10 minutes at 55° C.followed by immediately cooling to ˜32° C.). The partially digestedsample is then boiled and transferred to ice. This is followed byTerminal Deoxynucleotidyl (TdT) tailing with dTTP at 37° C. for 30minutes. The sample is boiled again after completion of the tailingreaction, followed by a ligation reaction wherein capture sequences,complementary to the poly T tail and labeled with a fluorescent dye,such as Cy3/green and Cy5/red, are ligated onto the strands. If fetalDNA is labeled with Cy3 then the maternal DNA is labeled with FITC, orvice versa. The ligation reaction is allowed to proceed for 30 minutesat room temperature before it is stopped by the addition of 0.5M EDTA.Labeled DNAs are then purified from the reaction components using acleanup kit, such as the Zymo DNA Clean and Concentration kit. Purifiedtagged DNAs are resuspended in a mixture containing 2× hybridizationbuffer, which contains LNA dT blocker, calf thymus DNA, and nucleasefree water. The mixture is vortexed at 14,000 RPM for one minute afterthe tagged DNA is added, then it is incubated at 95° C.-100° C. for 10minutes. The tagged. DNA hybridization mixture containing both labeled.DNAs is then incubated on a glass hybridization slide, which has beenprepared with human bacterial artificial chromsomes (BAC), such as the32K array set. BAC clones covering at least 98% of the human genome areavailable from BACPAC Resources, Oakland Calif.

The slide is then incubated overnight (˜16 hours) in a dark humidifiedchamber at 52° C. The slide is then washed using multiple posthybridization washed. The BAC microarray is then imaged using anepifluorescence microscope and a CCD camera interfaced to a computer.Analysis of the microarray images is performed using the GenePix Pro 4.0software (Axon Instruments, Foster City Calif.). For each spot themedian pixel intensity minus the median local background for both dyesis used to obtain a test over reference gene copy number ratio. Datanormalization is performed per array sub-grid using lowest curve fittingwith a smoothing factor of 0.33. To identify imbalances the MATLABtoolbox CGH plotter is applied, using moving mean average over threeclones and limits of log 2>0.2. Classification as gain or loss is basedon (1) identification as such by the CGH plotter and (2) visualinspection of the log 2 ratios. In general, log 2 ratios>0.5 in at leastfour adjacent clones will be considered to be deviating. Ratios of0.5-1.0 will be classified as duplications/hemizygous deletions;whereas, ratios>1 will be classified as amplifications/homozygousdeletions. All normalizations and analyses are carried out usinganalysis software, such as the BioArray Software Environment database.Regions of the genome that are either gained or lost in the fetal cellsare indicated by the fluorescence intensity ratio profiles. Thus, in asingle hybridization it is possible to screen the vast majority ofchromosomal sites that may contain genes that are either deleted oramplified in the fetal cells

The sensitivity of CGH in detecting gains and losses of DNA sequences isapproximately 0.2-20 Mb. For example, a loss of a 200 kb region shouldbe detectable under optimal hybridization conditions. Prior to CGHhybridization, DNA can be universally amplified using degenerateoligonucleotide-primed PCR (DOP-PCR), which allows the analysis of, forexample, rare fetal cell samples. The latter technique requires a PCRpre-amplification step.

Primers used for DOP-PCR have defined sequences at the 5′ end and at the3′ end, but have a random hexamer sequence between the two defined ends.The random hexamer sequence displays all possible combinations of thenatural nucleotides A, G, C, and T. DOP-PCR primers are annealed at lowstringency to the denatured template DNA and hybridize statistically toprimer binding sites. The distance between primer binding sites can becontrolled by the length of the defined sequence at the 3′ end and thestringency of the annealing conditions. The first five cycles of theDOP-PCR thermal cycle consist of low stringency annealing, followed by aslow temperature increase to the elongation temperature, and primerelongation. The next thirty-five cycles use a more stringent (higher)annealing temperature. Under the more stringent conditions the materialwhich was generated in the first five cycles is amplifiedpreferentially, since the complete primer sequence created at theamplicon termini is required for annealing. DOP-PCR amplificationideally results in a smear of DNA fragments that are visible on anagarose gel stained with ethidium bromide. These fragments can bedirectly labelled by ligating capture sequences, complementary to theprimer sequences and labeled with a fluorescent dye, such as Cy3/greenand Cy5/red. Alternatively the primers can be labelled with a florescentdye, in a manner that minimizes steric hindrance, prior to theamplification step.

Example 11 Isolation of Epithelial Cells from Blood

Microfluidic devices of the invention were designed by computer-aideddesign (CAD) and microfabricated by photolithography. A two-step processwas developed in which a blood sample is first debulked to remove thelarge population of small cells, and then the rare target epithelialcells target cells are recovered by immunoaffinity capture. The deviceswere defined by photolithography and etched into a silicon substratebased on the CAD-generated design. The cell enrichment module, which isapproximately the size of a standard microscope slide, contains 14parallel sample processing sections and associated sample handlingchannels that connect to common sample and buffer inlets and product andwaste outlets. Each section contains an array of microfabricatedobstacles that is optimized to enrich the target cell type byhydrodynamic size via displacement of the larger cells into the productstream. In this example, the microchip was designed to separate redblood cells (RBCs) and platelets from the larger leukocytes and CTCs.Enriched populations of target cells were recovered from whole bloodpassed through the device. Performance of the cell enrichment microchipwas evaluated by separating RBCs and platelets from white blood cells(WBCs) in normal whole blood (FIG. 18). In cancer patients, CTCs arefound in the larger WBC fraction. Blood was minimally diluted (30%), anda 6 ml sample was processed at a flow rate of up to 6 ml/hr. The productand waste stream were evaluated in a Coulter Model “A^(C)-T diff”clinical blood analyzer, which automatically distinguishes, sizes, andcounts different blood cell populations. The enrichment chip achievedseparation of RBCs from WBCs, in which the WBC fraction had >99%retention of nucleated cells, >99% depletion of RBCs, and >97% depletionof platelets. Representative histograms of these cell fractions areshown in FIG. 19. Routine cytology confirmed the high degree ofenrichment of the WBC and RBC fractions (FIG. 20).

Next, epithelial cells were recovered by affinity capture in amicrofluidic module that is functionalized with immobilized antibody. Acapture module with a single chamber containing a regular array ofantibody-coated microfabricated obstacles was designed. These obstaclesare disposed to maximize cell capture by increasing the capture areaapproximately four-fold, and by slowing the flow of cells under laminarflow adjacent to the obstacles to increase the contact time between thecells and the immobilized antibody. The capture modules may be operatedunder conditions of relatively high flow rate but low shear to protectcells against damage. The surface of the capture module wasfunctionalized by sequential treatment with 10% silane, 0.5%gluteraldehyde, and avidin, followed by biotinylated anti-EpCAM. Activesites were blocked with 3% bovine serum albumin in PBS, quenched withdilute Iris HCl, and stabilized with dilute L-histidine. Modules werewashed in PBS after each stage and finally dried and stored at roomtemperature. Capture performance was measured with the human advancedlung cancer cell line NCI—H1650 (ATCC Number CRL-5883). This cell linehas a heterozygous 15 bp in-frame deletion in exon 19 of EGFR thatrenders it susceptible to gefitinib. Cells from confluent cultures wereharvested with trypsin, stained with the vital dye Cell Tracker Orange(CMRA reagent, Molecular Probes, Eugene, Oreg.), resuspended in freshwhole blood, and fractionated in the microfluidic chip at various flowrates. In these initial feasibility experiments, cell suspensions wereprocessed directly in the capture modules without prior fractionation inthe cell enrichment module to debulk the red blood cells; hence, thesample stream contained normal blood red cells and leukocytes as well astumor cells. After the cells were processed in the capture module, thedevice was washed with buffer at a higher flow rate (3 ml/hr) to removethe nonspecifically bound cells. The adhesive top was removed and theadherent cells were fixed on the chip with paraformaldehyde and observedby fluorescence microscopy. Cell recovery was calculated fromhemacytometer counts; representative capture results are shown in Table4. Initial yields in reconstitution studies with unfractionated bloodwere greater than 60% with less than 5% of non-specific binding.

TABLE 4 Run Avg. flow Length of No. cells No. cells number rate runprocessed captured Yield 1 3.0 1 hr 150.000 38.012 25% 2 1.5 2 hr150.000 30.000/ml 60% 3 1.08 2 hr 106.000 66.661 64% 4 1.21 2 hr 121.00075.491 62%

Next, NCI—H1650 cells that were spiked into whole blood and recovered bysize fractionation and affinity capture as described above weresuccessfully analyzed in situ. In a trial run to distinguish epithelialcells from leukocytes, 0.5 ml of a stock solution of fluorescein-labeledCD45 pan-leukocyte monoclonal antibody were passed into the capturemodule and incubated at room temperature for 30 minutes. The module waswashed with buffer to remove unbound antibody, and the cells were fixedon the chip with 1% paraformaldehyde and observed by fluorescencemicroscopy. As shown in FIG. 21 the epithelial cells were bound to theobstacles and floor of the capture module. Background staining of theflow passages with CD45 pan-leukocyte antibody is visible, as areseveral stained leukocytes, apparently because of a low level ofnon-specific capture.

Example 12 Method for Detection of EGFR Mutations

A blood sample from a cancer patient is processed and analyzed using thedevices and methods of the invention, e.g., those of Example 11,resulting in an enriched sample of epithelial cells containing CTCs.This sample is then analyzed to identify potential EGFR mutations. Themethod permits both identification of known, clinically relevant EGFRmutations as well as discovery of novel mutations. An overview of thisprocess is shown in FIG. 22.

Below is an outline of the strategy for detection and confirmation ofEGFR mutations:

1) Sequence CTC EGFR mRNA

-   -   a) Purify CTCs from blood sample;    -   b) Purify total RNA from CTCs;    -   c) Convert RNA to cDNA using reverse transcriptase;    -   d) Use resultant cDNA to perform first and second PCR reactions        for generating sequencing templates; and    -   e) Purify the nested PCR amplicon and use as a sequencing        template to sequence EGFR exons 18-21.

2) Confirm. RNA sequence using CTC genomic DNA

-   -   a) Purify CTCs from blood sample;    -   b) Purify genomic DNA (gDNA) from CTCs;    -   c) Amplify exons 18, 19, 20, and/or 21 via PCR reactions; and    -   d) Use the resulting PCR amplicon(s) in real-time quantitative        allele-specific PCR reactions in order to confirm III the        sequence of mutations discovered via RNA sequencing.

Further details for each step outlined above are as follows:

1) Sequence CTC EGFR mRNA

a) Purify CTCs from blood sample. CTCs are isolated using any of thesize-based enrichment and/or affinity purification devices of theinvention.

b) Purify total RNA from CTCs. Total RNA is then purified from isolatedCTC populations using, e.g., the Qiagen Micro RNeasy kit, or a similartotal RNA purification protocol from another manufacturer;alternatively, standard RNA purification protocols such as guanidiumisothiocyanate homogenization followed by phenol/chloroform extractionand ethanol precipitation may be used.

c) Convert RNA to cDNA using reverse transcriptase. cDNA reactions arecarried out based on the protocols of the supplier of reversetranscriptase. Typically, the amount of input RNA into the cDNAreactions is in the range of 10 picograms (pg) to 2 micrograms (μg)total RNA. First-strand DNA synthesis is carried out by hybridizingrandom 7 mer DNA primers, or oligo-dT primers, or gene-specific primers,to RNA templates at 65° C. followed by snap-chilling on ice. cDNAsynthesis is initiated by the addition of iScript Reverse Transcriptase(BioRad) or SuperScript Reverse Transcriptase (Invitrogen) or a reversetranscriptase from another commercial vendor along with the appropriateenzyme reaction buffer. For iScript, reverse transcriptase reactions arecarried out at 42° C. for 30-45 minutes, followed by enzyme inactivationfor 5 minutes at 85° C. cDNA is stored at −20° C. until use or usedimmediately in PCR reactions. Typically, cDNA reactions are carried outin a final volume of 20 μl, and 10% (2 μl) of the resultant cDNA is usedin subsequent PCR reactions.

d) Use resultant cDNA to perform first and second PCR reactions forgenerating sequencing templates. cDNA from the reverse transcriptasereactions is mixed with DNA primers specific for the region of interest(FIG. 23). See Table 5 for sets of primers that may be used foramplification of exons 18-21. In Table 5, primer set M13(+)/M12(−) isinternal to primer set M11(+)/M14(−). Thus primers M13(+) and M12(−) maybe used in the nested round of amplification, if primers M11(+) andM14(−) were used in the first round of expansion. Similarly, primer setM11(+)/M14(−) is internal to primer set M15(+)/M16(−), and primer setM23H/M24(−) is internal to primer set M21(+)/M22(−). Hot Start PCRreactions are performed using Qiagen Hot-Star Taq Polymerase kit, orApplied Biosystems HotStart TaqMan polymerase, or other Hot Startthermostable polymerase, or without a hot start using Promega GoTaqGreen Taq Polymerase master mix, TaqMan DNA polymerase, or otherthermostable DNA polymerase. Typically, reaction volumes are 50 μl,nucleotide triphosphates are present at a final concentration of 200 μMfor each nucleotide, MgCl₂ is present at a final concentration of 1-4mM, and oligo primers are at a final concentration of 0.5 μM. Hot startprotocols begin with a 10-15 minute incubation at 95° C., followed by 40cycles of 94° C. for one minute (denaturation), 52° C. for one minute(annealing), and 72° C. for one minute (extension). A 10 minute terminalextension at 72° C. is performed before samples are stored at 4° C.until they are either used as template in the second (nested) round ofPCRs, or purified using QiaQuick Spin Columns (Qiagen) prior tosequencing. If a hot-start protocol is not used, the initial incubationat 95° C. is omitted. If a PCR product is to be used in a second roundof PCRs, 2 μl (4%) of the initial PCR product is used as template in thesecond round reactions, and the identical reagent concentrations andcycling parameters are used.

TABLE 5 Primer Sets for expanding EGFR mRNA around Exons 18-21 SEQ cDNAAmplicon Name ID NO Sequence (5′ to 3′) Coordinates Size NXK-M11(+) 111TTGCTGCTGGTGGTGGC (+) 1966-1982 813 NXK-M14(−) 112 CAGGGATTCCGTCATATGGC(−) 2778-2759 NXK-M13(+) 113 GATCGGCCTCTTCATGCG (+) 1989-2006 747NXK M12(−) 114 GATCCAAAGGTCATCAACTCCC (−) 2735-2714 NXK-M15(+) 115GCTGTCCAACGAATGGGC (+) 1904-1921 894 NXK-M16(−) 116 GGCGTTCTCCTTTCTCCAGG(−) 2797-2778 NXK-M21(+) 117 ATGCACTGGGCCAGGTCTT (+) 1881-1899 944NXK-M22(−) 118 CGATGGTACATATGGGTGGCT (−) 2824-2804 NXK-M23(+) 119AGGCTGTCCAACGAATGGG (+) 1902-1920 904 NXK-M24(−) 120 CTGAGGGAGGCGTTCTCCT(−) 2805-2787

e) Purify the nested PCR amplicon and use as a sequencing template tosequence EGFR exons 18-21. Sequencing is performed by ABI automatedfluorescent sequencing machines and fluorescence-labeled DNA sequencingladders generated via Sanger-style sequencing reactions usingfluorescent dideoxynucleotide mixtures. PCR products are purified usingQiagen QuickSpin columns, the Agencourt AMPure PCR Purification System,or PCR product purification kits obtained from other vendors. After PCRproducts are purified, the nucleotide concentration and purity isdetermined with a Nanodrop 7000 spectrophotometer, and the PCR productconcentration is brought to a concentration of 25 ng/μl, As a qualitycontrol measure, only PCR products that have a UV-light absorbance ratio(A₂₆₀/A₂₈₀) greater than 1.8 are used for sequencing. Sequencing primersare brought to a concentration of 3.2 pmol/μl.

2) Confirm RNA sequence using CTC genomic DNA

a) Purify CTCs from blood sample. As above, CTCs are isolated using anyof the size-based enrichment and/or affinity purification devices of theinvention.

b) Purify genomic DNA (gDNA) from CTCs. Genomic DNA is purified usingthe Qiagen DNeasy Mini kit, the Invitrogen ChargeSwitch gDNA kit, oranother commercial kit, or via the following protocol:

1. Cell pellets are either lysed fresh or stored at −80° C. and arethawed immediately before lysis.

2. Add 500 μl 50 mM Tris pH 7.9/100 mM EDTA/0.5% SDS (TES buffer).

3. Add 12.5 μl Proteinase K (IBI5406, 20 mg/ml), generating a final[ProtK]=0.5 mg/ml.

4. Incubate at 55° C. overnight in rotating incubator.

5. Add 20 μl of RNase cocktail (500 U/ml RNase A 20,000 U/ml RNase T1,Ambion #2288) and incubate four hours at 37° C.

6. Extract with Phenol (Kodak, Iris pH 8 equilibrated), shake to mix,spin 5 min. in tabletop centrifuge.

7. Transfer aqueous phase to fresh tube.

8. Extract with Phenol/Chloroform/isoamyl alcohol (EMD, 25:24:1 ratio,Tris pH 8 equilibrated), shake to mix, spin five minutes in tabletopcentrifuge.

9. Add 50 μl 3M NaOAc pH=6.

10. Add 500 μl EtOH.

11. Shake to mix. Strings of precipitated DNA may be visible. Ifanticipated DNA concentration is very low, add carrier nucleotide(usually yeast tRNA).

12. Spin one minute at max speed in tabletop centrifuge.

13. Remove supernatant.

14. Add 500 μl 70% EtOH, Room Temperature (RT)

15. Shake to mix.

16. Spin one minute at max speed in tabletop centrifuge.

17. Air dry 10-20 minutes before adding TE.

18. Resuspend in 400 μl TE. Incubate at 65° C. for 10 minutes, thenleave at RT overnight before quantitation on Nanodrop.

Amplify exons 18, 19, 20, and/or 21 via PCR reactions. Hot start nestedPCR amplification is carried out as described above in step 1d, exceptthat there is no nested round of amplification. The initial. PCR stepmay be stopped during the log phase in order to minimize possible lossof allele-specific information during amplification. The primer setsused for expansion of EGFR exons 18-21 are listed in Table 6 (see alsoPaez et al., Science 304:1497-1500 (Supplementary Material) (2004)).

TABLE 6 Primer sets for expanding EGER genomic DNA SEQ ID Amplicon NameNO Sequence (5′ to 3′) Exon Size NXK-ex18.1(+) 121TCAGAGCCTGTGTTTCTACCAA 18 534 NXK-ex18.2(−) 122 TGGTCTCACAGGACCACTGATT18 NXK-ex18.3(+) 123 TCCAAATGAGCTGGCAAGTG 18 397 NXK-ex18.4(−) 124TCCCAAACACTCAGTGAAACAAA 18 NXK-ex19.1(+) 125 AAATAATCAGTGTGATTCGTGGAG 19495 NXK-ex19.2(−) 126 GAGGCCAGTGCTGTCTCTAAGG 19 NXK-ex19.3(+) 127GTGCATCGCTGGTAACATCC 19 298 NXK-ext9.4(−) 128 TGTGGAGATGAGCAGGGTCT 19NXK-ex20.1(+) 129 ACTTCACAGCCCTGCGTAAAC 20 555 NXK-ex20.2(−) 130ATGGGACAGGCACTGATTTGT 20 NXK-ex20.3(+) 131 ATCGCATTCATGCGTCTTCA 20 379NXK-ex20.4(−) 132 ATCCCCATGGCAAACTCTTG 20 NXK-ex21.1(+) 133GCAGCGGGTTACATCTTCTTTC 21 526 NXK-ex21.2(−) 134 CAGCTCTGGCTCACACTACCAG21 NXK-ex21.3(+) 135 GCAGCGGGTTACATCTTCTTTC 21 349 NXK-ex21.4(−) 136CATCCTCCCCTGCATGTGT 21

d) Use the resulting PCR amplicon(s) in real-time quantitativeallele-specific PCR reactions in order to confirm the sequence ofmutations discovered via RNA sequencing. An aliquot of the PCR ampliconsis used as template in a multiplexed allele-specific quantitative PCRreaction using TaqMan PCR 5′ Nuclease assays with an Applied Biosystemsmodel 7500 Real Time PCR machine (FIG. 24). This round of PCR amplifiessubregions of the initial PCR product specific to each mutation ofinterest. Given the very high sensitivity of Real Time PCR, it ispossible to obtain complete information on the mutation status of theEGFR gene even if as few as 10 CTCs are isolated. Real Time PCR providesquantification of allelic sequences over 8 logs of input DNAconcentrations; thus, even heterozygous mutations in impure populationsare easily detected using this method.

Probe and primer sets are designed for all known mutations that affectgefitinib responsiveness in NSCLC patients, including over 40 suchsomatic mutations, including point mutations, deletions, and insertions,that have been reported in the medical literature. For illustrativepurposes, examples of primer and probe sets for five of the pointmutations are listed in Table 7. In general, oligonucleotides may bedesigned using the primer optimization software program Primer Express(Applied Biosystems), with hybridization conditions optimized todistinguish the wild type EGFR DNA sequence from mutant alleles. EGFRgenomic DNA amplified from lung cancer cell lines that are known tocarry EGFR mutations, such as H358 (wild type), H1650 (15-bp deletion,Δ2235-2249), and H1975 (two point mutations, 2369 C→T, 2573 T→+G), isused to optimize the allele-specific Real Time PCR reactions. Using theTaqMan 5′ nuclease assay, allele-specific labeled probes specific forwild type sequence or for known EGFR mutations are developed. Theoligonucleotides are designed to have melting temperatures that easilydistinguish a match from a mismatch, and the Real. Time PCR conditionsare optimized to distinguish wild type and mutant alleles. All Real TimePCR reactions are carried out in triplicate.

Initially, labeled probes containing wild type sequence are multiplexedin the same reaction with a single mutant probe. Expressing the resultsas a ratio of one mutant allele sequence versus wild type sequence mayidentify samples containing or lacking a given mutation. Afterconditions are optimized for a given probe set, it is then possible tomultiplex probes for all of the mutant alleles within a given exonwithin the same Real Time PCR assay, increasing the ease of use of thisanalytical tool in clinical settings.

A unique probe is designed for each wild type allele and mutant allelesequence. Wild-type sequences are marked with the fluorescent dye VIC atthe 5′ end, and mutant sequences with the fluorophore FAM. Afluorescence quencher and Minor Groove Binding moiety are attached tothe 3′ ends of the probes. ROX is used as a passive reference dye fornormalization purposes. A standard curve is generated for wild typesequences and is used for relative quantitation. Precise quantitation ofmutant signal is not required, as the input cell population is ofunknown, and varying, purity. The assay is set up as described by ABIproduct literature, and the presence of a mutation is confirmed when thesignal from a mutant allele probe rises above the background level offluorescence (FIG. 25), and this threshold cycle gives the relativefrequency of the mutant allele in the input sample.

TABLE 7 Probes and Primers for Allele-Specific qPCR SEQ Sequence (5′to 3′, ID mutated position cDNA Name NO in bold) Coordinates DescriptionMutation NXK-M01 137 CCGCAGCATGTCAAGATCAC (+) 2542- (+) primer L858R2561 NXK-M02 138 TCCTTCTGCATGGTATTCTTTCTCT (−) 2619- (−) primer 2595Pwt-L858R 139 VIC-TTTGGGCTGGCCAA-MGB (+) 2566- WT allele 2579 probePmut-L858R 140 FAM-TTTTGGGCGGGCCA-MGB (+) 2566- Mutant 2579 allele probeNXK-M03 141 ATGGCCAGCGTGGACAA (+) 2296- (+) primer T790M 2312 NXK-M04142 AGCAGGTACTGGGAGCCAATATT (−) 2444- (−) primer 2422 Pwt-T790M 143VIC-ATGAGCTGCGTGATGA-MGB (−) 2378- WT allele 2363 probe Pmut- 144FAM-ATGAGCTGCATGATGA-MGB (−) 2378- Mutant T790M 2363 allele probeNXIK-M05 145 GCCTCTTACACCCAGTGGAGAA (+) 2070- (+) primer G719S,C 2091NXK-M06 146 TTCTGGGATCCAGAGTCCCTTA (−) 2202- (−) primer 2181 Pwt-G7I9SC147 VIC-ACCGGAGCCCAGCA-MGB (−) 2163- WT allele 2150 probe Pmut-G719S 148FAM-ACCGGAGCTCAGCA-MGB (−) 2163- Mutant 2150 allele probe Pmut-G719C 149FAM-ACCGGAGCACAGCA-MGB (−) 2163- Mutant 2150 allele probe NXK-M09 150TCGCAAAGGGCATGAACTACT (+) 2462- (+) primer H835L 2482 NXK-M10 151ATCTTGACATGCTGCGGTGTT (−) 2558- (−) primer 2538 Pwt-H835L 152VIC-TTGGTGCACCGCGA-MGB (+) 2498- WT allele 2511 probe Pmut-H835L 153FAM-TGGTGCTCCGCGAC-MGB (+) 2498- Mutant 2511 allele probe

Example 13 Absence of EGFR Expression in Leukocytes

The protocol of Example 10 would be most useful if EGFR were expressedin target cancer cells but not in background leukocytes. To test whetherEGFR mRNA is present in leukocytes, several PCR experiments wereperformed. Four sets of primers, shown in Table 8, were designed toamplify four corresponding genes:

1) BCKDK (branched-chain a-ketoacid dehydrogenase complex kinase)—a“housekeeping” gene expressed in all types of cells, a positive controlfor both leukocytes and tumor cells;

2) CD45—specifically expressed in leukocytes, a positive control forleukocytes and a negative control for tumor cells;

3) EpCaM—specifically expressed in epithelial cells, a negative controlfor leukocytes and a positive control for tumor cells; and

4) EGFR—the target mRNA to be examined.

TABLE 8 SEQ ID Amplicon Name NO Sequence (5′ to 3′) Description SizeBCKD_1 154 AGTCAGGACCCATGCACGG BCKDK (+) primer 273 BCKD_2 155ACCCAAGATGCAGCAGTGTG BCKDK (−) primer CD_1 156 GATGTCCTCCTTGTTCTACTCCD45 (+) primer 263 CD_2 157 TACAGGGAATAATCGAGCATGC CD45 (−) primerEpCAM_1 158 GAAGGGAAATAGCAAATGGACA EpCAM (+) primer 222 EpCAM_2 159CGATGGAGTCCAAGTTCTGG EpCAM (−) primer EGFR_1 160 AGCACTTACAGCTCTGGCCAEGFR (+) primer 371 EGFR 2 161 GACTGAACATAACTGTAGGCTG EGER (−) primer

Total RNAs of approximately 9×10⁶ leukocytes isolated using a cellenrichment device of the invention (cutoff size 4 μm) and 5×10⁶ H1650cells were isolated by using RNeasy mini kit (Qiagen). Two micrograms oftotal RNAs from leukocytes and H1650 cells were reverse transcribed toobtain first strand cDNAs using 100 pmol random hexamer (Roche) and 200U Superscript II (Invitrogen) in a 20 μl reaction. The subsequent PCRwas carried out using 0.5 μl of the first strand cDNA reaction and 10pmol of forward and reverse primers in total 25 μl of mixture. The PCRwas run for 40 cycles of 95° C. for 20 seconds, 56° C. for 20 seconds,and 70° C. for 30 seconds. The amplified products were separated on a 1%agarose gel. As shown in FIG. 26A, BCKDK was found to be expressed inboth leukocytes and H1650 cells; CD45 was expressed only in leukocytes;and both EpCAM and EGFR were expressed only in H1650 cells. Theseresults, which are fully consistent with the profile of EGFR expressionshown in FIG. 26B, confirmed that EGFR is a particularly useful targetfor assaying mixtures of cells that include both leukocytes and cancercells, because only the cancer cells will be expected to produce asignal.

1.-57. (canceled)
 58. A method for determining a fetal aneuploidyconsisting of: obtaining a sample other than amniotic fluid sample froma pregnant female; enriching one or more fetal cells from said sampleusing size-based separation, wherein said size-based separation isperformed by flowing said sample or a fraction thereof through an arrayof obstacles that selectively directs cells larger than a predeterminedsize to a first outlet and cells smaller than said predetermined size toa second outlet, and wherein said array of obstacles for size-basedseparation is fluidly coupled with a second array of obstacles, whereinat least a portion of said obstacles of said second array are coatedwith binding moieties that specifically bind to one or more cellpopulations in said sample; obtaining one or more nucleic acid moleculesfrom said enriched fetal cells; amplifying said one or more nucleic acidmolecules; and analyzing said one of more amplified nucleic acidmolecules by mass spectrometry.